Bisfluoroalkyl-1,4-benzodiazepinone compounds

ABSTRACT

Disclosed are compounds of Formula (I) or prodrugs thereof; 
     
       
         
         
             
             
         
       
     
     wherein: R 1  is —CH 2 CF 3  or —CH 2 CH 2 CF 3 ; R 2  is —CH 2 CF 3 , —CH 2 CH 2 CF 3 , or —CH 2 CH 2 CH 2 CF 3 ; R 3  is H or —CH 3 ; each R a  is independently F, Cl, —CN, —OCH 3 , and/or —NHCH 2 CH 2 OCH 3 ; and z is zero, 1, or 2. Also disclosed are methods of using such compounds to inhibit the Notch receptor, and pharmaceutical compositions comprising such compounds. These compounds are useful in treating, preventing, or slowing the progression of diseases or disorders in a variety of therapeutic areas, such as cancer.

The present invention generally relates to benzodiazepinone compoundsuseful as Notch inhibitors. The invention further pertains topharmaceutical compositions comprising at least one compound accordingto the invention that is useful for the treatment of conditions relatedto the Notch pathway, such as cancer and other proliferative diseases.

Notch signaling has been implicated in a variety of cellular processes,such as cell fate specification, differentiation, proliferation,apoptosis, and angiogenesis. (Bray, Nature Reviews Molecular CellBiology, 7:678-689 (2006); Fortini, Developmental Cell 16:633-647(2009)). The Notch proteins are single-pass heterodimeric transmembranemolecules. The Notch family includes 4 receptors, NOTCH 1-4, whichbecome activated upon binding to ligands from the DSL family (Delta-like1, 3, 4 and Jagged 1 and 2).

The activation and maturation of NOTCH requires a series of processingsteps, including a proteolytic cleavage step mediated by gammasecretase, a multiprotein complex containing Presenilin 1 or Presenilin2, nicastrin, APH1, and PEN2. Once NOTCH is cleaved, NOTCH intracellulardomain (NICD) is released from the membrane. The released NICDtranslocates to the nucleus, where it functions as a transcriptionalactivator in concert with CSL family members (RBPSUH, “suppressor ofhairless”, and LAG1). NOTCH target genes include HES family members,such as HES-1. HES-1 functions as transcriptional repressors of genessuch as HERP1 (also known as HEY2), HERP2 (also known as HEY1), andHATH1 (also known as ATOH1).

The aberrant activation of the Notch pathway contributes totumorigenesis. Activation of Notch signaling has been implicated in thepathogenesis of various solid tumors including ovarian, pancreatic, aswell as breast cancer and hematologic tumors such as leukemias,lymphomas, and multiple myeloma. The role of Notch inhibition and itsutility in the treatment of various solid and hematological tumors aredescribed in Miele, L. et al., Current Cancer Drug Targets, 6:313-323(2006); Bolos, V. et al., Endocrine Reviews, 28:339-363 (2007); Shih,I.-M. et al., Cancer Research, 67:1879-1882 (2007); Yamaguchi, N. etal., Cancer Research, 68:1881-1888 (2008); Miele, L., Expert ReviewAnti-cancer Therapy, 8:1197-1201 (2008); Purow, B., CurrentPharmaceutical Biotechnology, 10:154-160 (2009); Nefedova, Y. et al.,Drug Resistance Updates, 11:210-218 (2008); Dufraine, J. et al.,Oncogene, 27:5132-5137 (2008); and Jun, H. T. et al., Drug DevelopmentResearch, 69:319-328 (2008).

There remains a need for compounds that are useful as Notch inhibitorsand that have sufficient metabolic stability to provide efficaciouslevels of drug exposure. Further, there remains a need for compoundsuseful as Notch inhibitors that can be orally or intravenouslyadministered to a patient.

U.S. Pat. No. 7,053,084 B1 discloses succinoylamino benzodiazepinecompounds useful for treating neurological disorders such as Alzheimer'sDisease. The reference discloses that these succinoylaminobenzodiazepine compounds inhibit gamma secretase activity and theprocessing of amyloid precursor protein linked to the formation ofneurological deposits of amyloid protein. The reference does notdisclose the use of these compounds in the treatment of proliferativediseases such as cancer.

Applicants have found potent compounds that have activity as Notchinhibitors and have sufficient metabolic stability to provideefficacious levels of drug exposure upon intravenous or oraladministration. These compounds are provided to be useful aspharmaceuticals with desirable stability, bioavailability, therapeuticindex, and toxicity values that are important to their drugability.

SUMMARY OF THE INVENTION

The present invention fills the foregoing need by providingbis(fluoroalkyl)-1,4-benzodiazepinone compounds that are useful asselective inhibitors of Notch signaling pathway, including prodrugsthereof.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier; and at least onecompound of Formula (I) or prodrugs thereof.

The present invention also provides a method of treating a disease ordisorder associated with the activity of the Notch receptor, the methodcomprising administering to a mammalian patient a compound of Formula(I) or pharmaceutically acceptable prodrugs thereof.

The present invention also provides processes and intermediates formaking the compounds of Formula (I) or prodrugs thereof.

The present invention also provides the compounds of Formula (I), orprodrugs thereof, for use in therapy.

The present invention also provides the use of the compounds of Formula(I), or prodrugs thereof, for the manufacture of a medicament for thetreatment of cancer.

The compounds of Formula (I) and compositions comprising the compoundsare Notch inhibitors may be used in treating, preventing or curingvarious Notch receptor-related conditions. Pharmaceutical compositionscomprising these compounds are useful in treating, preventing, orslowing the progression of diseases or disorders in a variety oftherapeutic areas, such as cancer.

These and other features of the invention will be set forth in expandedform as the disclosure continues.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated by reference to the accompanying drawingsdescribed below.

FIG. 1 shows the experimental (at approximately 25° C.) and thesimulated (at approximately 25° C.) PXRD patterns (CuKα λ=1.5418 Å) ofthe N-1 Form of the compound of Example 1.

FIG. 2 shows the experimental (at approximately 25° C.) and thesimulated (at approximately 25° C.) PXRD patterns (CuKα λ=1.5418 Å) ofthe A-2 Form of the compound of Example 1.

FIG. 3 shows the experimental (at approximately 25° C.) and thesimulated (at approximately 25° C.) PXRD patterns (CuKα λ=1.5418 Å) ofthe EA-3 Form of the compound of Example 1.

FIG. 4 shows the experimental (at approximately 25° C.) and thesimulated (at approximately −50° C.) PXRD patterns (CuKα λ=1.5418 Å) ofthe THF-2 Form of the compound of Example 1.

FIG. 5 shows the experimental (at approximately 25° C.) and thesimulated (at approximately 25° C.) PXRD patterns (CuKα λ=1.5418 Å) ofthe M2-1 Form of the compound of Example 2.

FIG. 6 shows the antitumor efficacy of Example 1 against TALL1 HumanT-cell acute lymphoblastic leukemia. Each symbol represents the mediantumor burden of a group of 8 mice. () Control; (▪) Example 1, 5mg/kg/adm, QD×3, IV.

FIG. 7 shows the in vivo antitumor activity of Example 2 in T-cell acutelymphoblastic leukemia cell line TALL1. Each symbol represents themedian tumor burden of a group of 8 mice. () control; (Δ) Example 2, 12mg/kg, QD×15; (▪) Example 2, 6 mg/kg, QD×15; ( ) Example 2, 3 mg/kg,QD×15; (▴) Example 2, 1.5 mg/kg, QD×15; (∘) Example 2, 0.75 mg/kg,QD×15.

FIG. 8 shows the in vivo antitumor activity of Example 2 in human breastcarcinoma cell line MDA-MB-157. Each symbol represents the median tumorburden of a group of 8 mice. (∘) control; (▴) Example 2, 24 mg/kg; (▪)Example 2, 18 mg/kg; (▪) Example 2, 12 mg/kg.

FIG. 9 shows the synergistic antitumor efficacy by combined chemotherapywith Example 1 and dasatinib in the ALL-SIL T-cell lymphoblasticleukemia. Each symbol represents the median tumor burden of a group of 8mice. () control; (♦) Example 1, 3.75 mg/kg/adm, QD×3, weekly for 7weeks, PO; (▪) dasatinib, 10 mg/kg/adm, QD×49, PO; (□) Example 1, 3.75mg/kg/adm, QD×3, weekly for 7 weeks, PO+dasatinib 10 mg/kg/adm, QD×49,PO. When administered on the same day, the two agents were given more orless simultaneously (Example 1 preceded dasatinib by less than 1 hr).

FIG. 10 shows the synergistic antitumor efficacy by combinedchemotherapy with Example 1 and dasatinib in the ALL-SIL T-celllymphoblastic leukemia. Each symbol represents the median tumor burdenof a group of 8 mice. () control; (♦) Example 1, 7.5 mg/kg/adm QD×3,weekly for 7 weeks, PO; (▪) dasatinib, 10 mg/kg/adm, QD×49, PO; (□)Example 1, 7.5 mg/kg/adm, QD×3, weekly for 7 weeks, PO+dasatinib 10mg/kg/adm, QD×49, PO. When administered on the same day, the two agentswere given more or less simultaneously (Example 1 preceded dasatinib byless than 1 hr).

FIG. 11 shows the synergistic antitumor efficacy by combinedchemotherapy with Example 1 and Paclitaxel in the MDA-MB-468 HumanBreast Carcinoma. Each symbol represents the median tumor burden of agroup of 8 mice. () control; (♦) Paclitaxel, 12 mg/kg/adm, Q7D×6, IV;(▴) Example 1, 3.75 mg/kg/adm, QD×3, weekly for 7 weeks, PO; (□)Combination of Example 1 and Paclitaxel.

FIG. 12 shows the synergistic antitumor efficacy by combinedchemotherapy with Example 1 and Paclitaxel in the MDA-MB-468 HumanBreast Carcinoma. Each symbol represents the median tumor burden of agroup of 8 mice. () control; (♦) Paclitaxel, 12 mg/kg/adm, Q7D×6, IV;(♦) Example 1, 7.5 mg/kg/adm, QD×3, weekly for 7 weeks, PO; (□)Combination of Example 1 and Paclitaxel.

FIG. 13 shows the synergistic antitumor efficacy by combinedchemotherapy with Example 1 and Tamoxifen in the MCF7 Human BreastCarcinoma. Each symbol represents the median tumor burden of a group of8 mice. () control; (▪) Tamoxifen, 20 mg/kg/adm, Q2D×12, IP; (♦)Example 1, 3.75 mg/kg/adm, QD×3, weekly for 3 weeks, PO; (□) Combinationof Example 1 and Tamoxifen.

FIG. 14 shows the synergistic antitumor efficacy by combinedchemotherapy with Example 1 and Tamoxifen in the MCF7 Human BreastCarcinoma. Each symbol represents the median tumor burden of a group of8 mice. () control; (▪) Tamoxifen, 20 mg/kg/adm, Q2D×12, IP; (♦)Example 1, 7.5 mg/kg/adm, QD×3, weekly for 3 weeks, PO; (□) Combinationof Example 1 and Tamoxifen.

FIG. 15 shows the synergistic antitumor efficacy by combinedchemotherapy with Example 1 and dexamethasone (Dexa) in human T-ALLleukemia xenografts HPB-ALL. Each symbol represents the median tumorburden of a group of 6-8 mice. () control; (♦) dexamethasone, 7.5mg/kg/adm, QD×14, IP; (Δ) Example 1, 3.75 mg/kg/adm, QD×3, weekly for 3weeks, PO; (□) Combination of Example 1 and dexamethasone.

FIG. 16 shows the synergistic antitumor efficacy by combinedchemotherapy with Example 1 and carboplatin in PA-1 human ovarianteratocarcinoma. Each symbol represents the median tumor burden of agroup of 8 mice. () control; (□) carboplatin, 90 mg/kg/adm, Q7D×3, IV;(Δ) Example 1, 1 mg/kg/adm, QD×21, PO; (♦) Combination of Example 1 andcarboplatin.

DETAILED DESCRIPTION

The first aspect of the present invention provides compounds of Formula(I):

or prodrugs thereof; wherein:

R₁ is —CH₂CF₃ or —CH₂CH₂CF₃;

R₂ is —CH₂CF₃, —CH₂CH₂CF₃, or —CH₂CH₂CH₂CF₃;

R₃ is H or —CH₃;

each R_(a) is independently F, Cl, —CN, —OCH₃, and/or —NHCH₂CH₂OCH₃; andz is zero, 1, or 2.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CF₃; and R₂, R₃, R_(a), and z are defined in the firstaspect. Included in this embodiment are compounds in which R₂ is —CH₂CF₃or —CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CH₂CF₃; and R₂, R₃, R_(a), and z are defined in thefirst aspect. Included in this embodiment are compounds in which R₂ is—CH₂CF₃ or —CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₂ is —CH₂CF₃; and R₁, R₃, R_(a), and z are defined in the firstaspect. Included in this embodiment are compounds in which R₁ is—CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₂ is —CH₂CH₂CF₃; and R₁, R₃, R_(a), and z are defined in thefirst aspect. Included in this embodiment are compounds in which R₁ is—CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₂ is —CH₂CH₂CH₂CF₃; and R₁, R₃, R_(a), and z are defined in thefirst aspect. Included in this embodiment are compounds in which R₁ is—CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₃ is H; and R₁, R₂, R_(a), and z are defined in the firstaspect. Included in this embodiment are compounds in which R₁ isdeuterium (D) or tritium (T). Also included in this embodiment arecompounds in which R₁ is —CH₂CH₂CF₃ and R₂ is —CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₃ is —CH₃; and R₁, R₂, R_(a), and z are defined in the firstaspect. R₃ includes methyl groups in which one or more hydrogen atomsare isotopically substituted with deuterium (D) and/or tritium (T). Inone example of this embodiment, R₃ is —CD₃. Also included in thisembodiment are compounds in which R₁ is —CH₂CH₂CF₃ and R₂ is —CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein z is 2 and each R_(a) is independently F, Cl, —CN, —OCH₃, and/or—NHCH₂CH₂OCH₃; and R₁, R₂, and R₃ are defined in the first aspect.Included in this embodiment are compounds in which R₁ is —CH₂CH₂CF₃ andR₂ is —CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein z is 1 and R_(a) is F, Cl, —CN, —OCH₃, or —NHCH₂CH₂OCH₃; and R₁,R₂, and R₃ are defined in the first aspect. Included in this embodimentare compounds in which R₁ is —CH₂CH₂CF₃ and R₂ is —CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein z is zero; and R₁, R₂, and R₃ are defined in the first aspect.Included in this embodiment are compounds in which R₁ is —CH₂CH₂CF₃ andR₂ is —CH₂CF₃ or —CH₂CH₂CF₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CF₃; R₂ is —CH₂CF₃; R₃ is H or —CH₃; and z is zero.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CF₃; R₂ is —CH₂CH₂CF₃; R₃ is H or —CH₃; and z is zero.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CF₃; R₂ is —CH₂CH₂CH₂CF₃; R₃ is H or —CH₃; and z iszero.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CH₂CF₃; R₂ is —CH₂CF₃; R₃ is H or —CH₃; and z is zero.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CH₂CF₃; R₂ is —CH₂CH₂CF₃; R₃ is H or —CH₃; and z iszero.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CH₂CF₃; R₂ is —CH₂CH₂CH₂CF₃; R₃ is H or —CH₃; and z iszero.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CF₃; R₂ is —CH₂CF₃; R₃ is H or —CH₃; z is 1; and R_(a)is F, Cl, —CN, —OCH₃, and/or —NHCH₂CH₂OCH₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CF₃; R₂ is —CH₂CH₂CF₃; R₃ is H or —CH₃; z is 1; andR_(a) is F, Cl, —CN, —OCH₃, and/or —NHCH₂CH₂OCH₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CF₃; R₂ is —CH₂CH₂CH₂CF₃; R₃ is H or —CH₃; z is 1; andR_(a) is F, Cl, —CN, —OCH₃, and/or —NHCH₂CH₂OCH₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CH₂CF₃; R₂ is —CH₂CF₃; R₃ is H or —CH₃; z is 1; andR_(a) is F, Cl, —CN, —OCH₃, and/or —NHCH₂CH₂OCH₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CH₂CF₃; R₂ is —CH₂CH₂CF₃; R₃ is H or —CH₃; z is 1; andR_(a) is F, Cl, —CN, —OCH₃, and/or —NHCH₂CH₂OCH₃.

One embodiment provides a compound of Formula (I) or prodrugs thereof,wherein R₁ is —CH₂CH₂CF₃; R₂ is —CH₂CH₂CH₂CF₃; R₃ is H or —CH₃; z is 1;and R_(a) is F, Cl, —CN, —OCH₃, and/or —NHCH₂CH₂OCH₃.

One embodiment provides a compound according to claim 1 or prodrugsthereof, selected from:

One embodiment provides a compound according to claim 1 or prodrugsthereof, selected from:

One embodiment provides a compound according to claim 1 or prodrugsthereof, selected from:

One embodiment provides a compound according to claim 1 or prodrugsthereof, selected from:

One embodiment provides a compound of Formula (I) selected from:(2R,3S)—N-((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(1);(2R,3S)—N-((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(2);(2R,3S)—N-((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2-(2,2,2-trifluoroethyl)-3-(3,3,3-trifluoropropyl)succinamide(3);(2R,3S)—N-((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-3-(2,2,2-trifluoroethyl)-2-(3,3,3-trifluoropropyl)succinamide(4);(2R,3S)—N-((3S)-1-(²H₃)methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(5);(2R,3S)—N-((3S)-7-chloro-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(6);(2R,3S)—N-((3S)-8-methoxy-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(7);(2R,3S)—N-((3S)-8-fluoro-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(8);(2R,3S)—N-((3S)-7-methoxy-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(9);(2R,3S)—N-((3S)-7-fluoro-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(10);(2R,3S)—N-((3S)-8-chloro-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(11);(2R,3S)—N-((3S)-9-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(12);(2R,3S)—N-((3S)-8-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(13);(2R,3S)—N-((3S)-7-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(14);(2R,3S)—N-((3S)-8-cyano-9-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(15);(2R,3S)—N-((3S)-8,9-dichloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(16);(2R,3S)—N-((3S)-9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(17);(2R,3S)—N-((3S)-9-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(18);(2R,3S)—N-((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-3-(4,4,4-trifluorobutyl)-2-(3,3,3-trifluoropropyl)succinamide(19);(2R,3S)—N1-((3S)-8-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-3-(4,4,4-trifluorobutyl)-2-(3,3,3-trifluoropropyl)succinamide(20); and(2R,3S)—N-((3S)-9-((2-methoxyethyl)amino)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(21); and prodrugs of one or more of the above compounds.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Thisinvention encompasses all combinations of the aspects and/or embodimentsof the invention noted herein. It is understood that any and allembodiments of the present invention may be taken in conjunction withany other embodiment or embodiments to describe addition moreembodiments. It is also to be understood that each individual element ofthe embodiments is meant to be combined with any and all other elementsfrom any embodiment to describe an additional embodiment.

DEFINITIONS

The features and advantages of the invention may be more readilyunderstood by those of ordinary skill in the art upon reading thefollowing detailed description. It is to be appreciated that certainfeatures of the invention that are, for clarity reasons, described aboveand below in the context of separate embodiments, may also be combinedto form a single embodiment. Conversely, various features of theinvention that are, for brevity reasons, described in the context of asingle embodiment, may also be combined so as to form sub-combinationsthereof. Embodiments identified herein as exemplary or preferred areintended to be illustrative and not limiting.

Unless specifically stated otherwise herein, references made in thesingular may also include the plural. For example, “a” and “an” mayrefer to either one, or one or more.

Unless otherwise indicated, any heteroatom with unsatisfied valences isassumed to have hydrogen atoms sufficient to satisfy the valences.

The definitions set forth herein take precedence over definitions setforth in any patent, patent application, and/or patent applicationpublication incorporated herein by reference.

Listed below are definitions of various terms used to describe thepresent invention. These definitions apply to the terms as they are usedthroughout the specification (unless they are otherwise limited inspecific instances) either individually or as part of a larger group.

Throughout the specification, groups and substituents thereof may bechosen by one skilled in the field to provide stable moieties andcompounds.

In accordance with a convention used in the art,

is used in structural formulas herein to depict the bond that is thepoint of attachment of the moiety or substituent to the core or backbonestructure.

The terms “halo” and “halogen,” as used herein, refer to F, Cl, Br, orI.

The term “alkyl” as used herein, refers to both branched and straightchain saturated aliphatic hydrocarbon groups containing, for example,from 1 to 12 carbon atoms, from 1 to 6 carbon atoms, and from 1 to 4carbon atoms. Examples of alkyl groups include, but are not limited to,methyl (Me), ethyl (Et), propyl (e.g., n-propyl and i-propyl), butyl(e.g., n-butyl, i-butyl, sec-butyl, and t-butyl), and pentyl (e.g.,n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylpentyl, 2-ethylbutyl,3-methylpentyl, and 4-methylpentyl. When numbers appear in a subscriptafter the symbol “C”, the subscript defines with more specificity thenumber of carbon atoms that a particular group may contain. For example,“C₁₋₆alkyl” denotes straight and branched chain alkyl groups with one tosix carbon atoms.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The compounds of Formula (I) can be provided as amorphous solids orcrystalline solids. Lyophilization can be employed to provide thecompounds of Formula (I) as a solid.

Any compound that can be converted in vivo to provide the bioactiveagent (i.e., the compound of Formula (I)) is a prodrug within the scopeand spirit of the invention.

Various forms of prodrugs are well known in the art and are describedin:

-   a) Wermuth, C. G. et al., The Practice of Medicinal Chemistry,    Chapter 31, Academic Press (1996);-   b) Bundgaard, H. ed., Design of Prodrugs, Elsevier (1985);-   c) Bundgaard, H., Chapter 5, “Design and Application of Prodrugs,”    Krosgaard-Larsen, P. et al., eds., A Textbook of Drug Design and    Development, pp. 113-191, Harwood Academic Publishers (1991); and-   d) Testa, B. et al., Hydrolysis in Drug and Prodrug Metabolism,    Wiley-VCH (2003).

In addition, compounds of Formula (I) are, subsequent to theirpreparation, preferably isolated and purified to obtain a compositioncontaining an amount by weight equal to or greater than 99% of acompound of Formula (I) (“substantially pure”), which is then used orformulated as described herein. Such “substantially pure” compounds ofFormula (I) are also contemplated herein as part of the presentinvention.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. The present invention is intended toembody stable compounds.

“Therapeutically effective amount” is intended to include an amount of acompound of the present invention alone or an amount of the combinationof compounds claimed or an amount of a compound of the present inventionin combination with other active ingredients effective to act as aninhibitor to a NOTCH receptor, or effective to treat or preventproliferative diseases such as cancer.

As used herein, “treating” or “treatment” cover the treatment of adisease-state in a mammal, particularly in a human, and include: (a)preventing the disease-state from occurring in a mammal, in particular,when such mammal is predisposed to the disease-state but has not yetbeen diagnosed as having it; (b) inhibiting the disease-state, i.e.,arresting its development; and/or (c) relieving the disease-state, i.e.,causing regression of the disease state.

The compounds of the present invention are intended to include allisotopes of atoms occurring in the present compounds. Isotopes includethose atoms having the same atomic number but different mass numbers. Byway of general example and without limitation, isotopes of hydrogeninclude deuterium (D) and tritium (T). Isotopes of carbon include ¹³Cand ¹⁴C. Isotopically-labeled compounds of the invention can generallybe prepared by conventional techniques known to those skilled in the artor by processes analogous to those described herein, using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

Crystal Forms of the Compound of Example 1

In one embodiment, the compound of Example 1

is provided as a crystalline material comprising one or more crystallineforms. Examples of suitable crystalline forms of the compound of Example1 include Forms N-1, A-2, and EA-3.

In one embodiment, the compound of Example 1 is provided as acrystalline material comprising the first crystalline form. A firstcrystalline form of the compound of Example 1 comprises a neatcrystalline form referred to herein as “Form N-1” or “N-1 Form”.

In one embodiment, the N-1 Form of the compound of Example 1 ischaracterized by unit cell parameters approximately equal to thefollowing:

Cell dimensions:

-   -   a=9.41 Å    -   b=17.74 Å    -   c=31.94 Å    -   α=90.0°    -   β=98.4°    -   γ=90.0°

Space group: P2₁

Molecules of Example 1/asymmetric unit: 4

Volume/Number of molecules in the unit cell=659 Å³

Density (calculated)=1.402 g/cm³,

wherein the unit cell parameters of Form N-1 are measured at atemperature of about −10° C.

In another embodiment, the N-1 Form of the compound of Example 1 ischaracterized by a simulated powder x-ray diffraction (PXRD) patternsubstantially in accordance with the pattern shown in FIG. 1 and/or byan observed PXRD pattern substantially in accordance with the patternshown in FIG. 1

In yet another embodiment, the N-1 Form of the compound of Example 1 ischaracterized by a PXRD pattern (CuKα λ=1.5418 Å at a temperature ofabout 25° C.) comprising four or more, preferably five or more, 2θvalues selected from: 5.7±0.2, 7.5±0.2, 10.3±0.2, 10.7±0.2, 15.2±0.2,16.8±0.2, 20.2±0.2, and 20.7±0.2, wherein the PXRD pattern of Form N-1is measured at a temperature of about 20° C.

In yet an even further embodiment, the N-1 Form of Example 1 ischaracterized by fractional atomic coordinates substantially as listedin Table 1.

TABLE 1 Fractional Atomic Coordinates of Form N-1 of Example 1Calculated at a Temperature of about 25° C.; Atomic Coordinates (×10⁴)and Equivalent Isotropic Displacement Parameters (Å² × 10³) x y z U(eq)*N(7) 10824(5) 7415(3) 1732(1) 39(1) N(3) 4985(5) 565(2) 1293(1) 33(1)O(3) 7152(4) 45(2) 1509(1) 45(1) N(8) 9981(6) 10557(3) 1329(2) 60(2)O(7) 12033(5) 10000(2) 1229(1) 55(1) O(6) 8697(5) 7992(2) 1768(1) 53(1)F(4) 11039(5) 8541(3) 79(1) 76(1) C(17) 5836(7) 8(3) 1469(1) 30(1) N(11)3979(5) 1500(3) 3735(1) 35(1) N(2) 5338(6) 2530(3) 994(1) 41(1) C(8)7012(6) 1237(3) 77(2) 35(1) N(15) 9277(5) 4606(3) 3230(1) 37(1) C(39)9990(7) 8020(3) 1739(2) 36(1) O(2) 3365(5) 1827(2) 1076(1) 49(1) N(6)10513(6) 5383(3) 1910(2) 46(1) C(41) 10028(6) 9204(3) 1300(2) 36(1)C(18) 5094(6) −692(3) 1616(2) 33(1) C(22) 5502(6) −1369(3) 1360(2) 34(1)C(23) 4828(7) −2081(3) 1501(2) 35(1) C(2) 6832(7) 2584(3) 978(2) 44(2)C(14) 5639(7) 1198(3) 1113(2) 37(1) C(36) 10130(7) 6682(3) 1698(2) 37(1)C(15) 4647(7) 1875(3) 1071(2) 37(1) O(14) 3524(5) −2130(3) 1484(2) 63(1)O(9) 5867(5) 1970(2) 3461(1) 46(1) C(64) 4608(7) 2008(3) 3517(2) 35(1)C(49) 7025(7) 893(4) 5004(2) 47(2) O(12) 7212(5) 3943(2) 3143(1) 57(1)F(5) 8960(5) 8082(2) 93(1) 73(1) C(33) 8030(8) 6274(4) −21(2) 55(2) N(5)9704(5) 6528(3) 1249(1) 36(1) C(42) 10783(8) 9965(4) 1287(2) 46(2) O(5)12472(5) 6155(3) 1945(1) 62(1) C(37) 11163(8) 6062(4) 1872(2) 46(2) F(7)9290(6) 2987(3) 5202(1) 94(2) C(65) 3707(6) 2688(3) 3341(2) 33(1) N(4)5716(6) −2636(3) 1628(2) 53(1) C(7) 6778(6) 1429(3) 518(2) 34(1) O(8)2612(5) 231(2) 3943(1) 53(1) O(11) 10584(6) 5864(3) 3001(1) 61(1) C(89)8963(6) 2807(3) 3658(2) 41(1) C(70) 4269(7) 3355(4) 4041(2) 47(2) N(13)8438(5) 5493(3) 3689(1) 37(1) C(20) 5588(7) −808(3) 2089(2) 38(1) C(85)9369(8) 5946(4) 3070(2) 46(2) N(14) 8611(7) 6593(3) 3016(2) 55(1) N(16)8948(7) 1437(3) 3656(2) 68(2) N(9) 5416(5) 1109(3) 4375(1) 37(1) C(71)4930(8) 4055(3) 4280(2) 51(2) N(12) 4314(6) 4592(3) 3189(2) 68(2) C(1)7546(7) 2068(3) 746(2) 41(2) O(13) 11084(5) 2050(3) 3796(2) 67(1) C(73)7471(7) 6206(4) 4209(2) 46(2) C(6) 9033(7) 2147(4) 744(2) 51(2) C(88)9391(7) 3239(3) 3270(2) 44(2) C(40) 10716(7) 8794(3) 1708(2) 41(1) C(54)7871(7) 381(4) 5261(2) 47(2) C(67) 3574(8) 4095(4) 3382(2) 51(2) F(6)8838(6) 3995(2) 4842(1) 84(1) C(79) 7137(9) 6649(4) 3054(2) 56(2) C(24)8991(7) 5283(3) 1841(2) 45(2) C(31) 8282(6) 5837(3) 703(2) 37(1) C(47)9570(7) 9167(4) 501(2) 46(2) C(63) 4839(6) 898(3) 3940(2) 34(1) C(90)9762(7) 2060(3) 3707(2) 45(2) C(55) 6443(6) 715(3) 4559(2) 39(1) C(68)3723(8) 2732(4) 2864(2) 60(2) C(59) 8345(10) −1056(5) 3924(2) 70(2)C(75) 7821(8) 5880(5) 4945(2) 68(2) C(32) 8425(7) 6417(3) 405(2) 50(2)O(10) 2312(5) 4203(3) 3420(2) 75(2) C(48) 9694(8) 8740(4) 112(2) 57(2)N(10) 4682(6) −430(3) 4071(1) 46(1) C(60) 9196(10) −525(5) 4132(3) 78(3)C(72) 7560(6) 5996(3) 3766(2) 38(1) C(11) 7228(8) 906(5) −774(2) 65(2)C(95) 9510(8) 3334(4) 4845(2) 56(2) C(86) 8534(6) 5301(3) 3249(2) 36(1)C(16) 4463(10) 3237(4) 980(2) 75(3) C(62) 3917(7) 205(4) 3981(2) 43(2)C(46) 10181(7) 8747(4) 901(2) 44(2) C(57) 6210(8) −445(3) 4106(2) 51(2)C(30) 8759(6) 5995(3) 1157(2) 35(1) C(66) 4345(6) 3404(3) 3568(2) 37(1)C(87) 8540(6) 3940(3) 3212(2) 40(1) C(93) 9371(7) 3266(3) 4063(2) 46(2)C(58) 6870(9) −1041(4) 3907(2) 60(2) F(2) 11777(9) 10008(4) 2879(2)151(3) C(74) 7871(7) 5699(4) 4528(2) 55(2) C(25) 8132(7) 5566(3) 1483(2)41(1) C(4) 9061(10) 3259(5) 1173(2) 76(2) C(26) 6645(7) 5450(3) 1443(2)48(2) C(12) 7727(9) 1590(5) −595(2) 66(2) C(56) 7085(7) 83(4) 4344(2)45(2) C(34) 7488(7) 5600(4) −167(2) 55(2) C(9) 6519(7) 574(4) −101(2)49(2) C(3) 7607(10) 3182(4) 1190(2) 66(2) C(81) 5158(10) 6468(4) 3438(2)76(2) C(45) 11099(16) 9317(7) 2895(3) 121(4) C(43) 10543(9) 9268(4)2103(2) 64(2) F(3) 9787(11) 9550(9) 2938(2) 245(7) C(53) 8327(8) 558(5)5683(2) 66(2) C(29) 8344(10) 4873(4) 2138(2) 61(2) C(5) 9774(10) 2734(5)964(2) 76(2) C(80) 6621(7) 6399(3) 3417(2) 53(2) C(94) 9050(7) 2877(4)4460(2) 51(2) C(38) 11435(9) 4768(4) 2102(2) 71(2) C(13) 7606(7) 1760(3)−179(2) 48(2) C(61) 3913(9) −1147(4) 4095(2) 71(2) C(52) 8010(10)1214(6) 5841(2) 79(2) C(10) 6633(9) 406(4) −526(2) 68(2) C(35) 7696(8)5160(4) 553(2) 57(2) C(84) 9357(12) 7262(4) 2869(3) 91(3) C(91) 9330(8)2785(4) 2864(2) 59(2) C(27) 6019(9) 5060(4) 1739(2) 69(2) C(44)11196(10) 8906(4) 2495(2) 78(2) C(69) 3191(8) 2018(4) 2628(2) 61(2)C(78) 7034(8) 6922(4) 4311(2) 64(2) C(83) 6199(12) 7012(5) 2740(3) 87(3)C(82) 4221(10) 6784(6) 3121(4) 100(3) C(51) 7187(10) 1748(5) 5591(2)79(2) C(76) 7428(9) 6592(6) 5047(2) 80(3) F(1) 11661(10) 8983(5) 3228(2)186(4) C(50) 6711(8) 1571(4) 5174(2) 58(2) C(77) 7057(9) 7114(5) 4734(3)85(3) N(1) 5875(5) 999(2) 684(1) 34(1) C(102) 5582(7) −1953(3) 630(2)49(2) C(101) 5084(7) −1277(3) 879(2) 44(2) C(100) 5214(10) −152(4)2344(2) 73(2) C(104) 7326(8) 5036(4) 115(2) 70(2) C(28) 6897(11) 4774(4)2086(3) 75(2) F(40) 9173(5) 9112(2) −241(1) 74(1) C(105) 8587(8) 41(4)4352(2) 60(2) C(124) 2922(10) 2104(6) 2165(3) 82(3) F(42) 4029(8)2397(5) 2012(2) 142(3) F(43) 1850(8) 2595(5) 2046(2) 134(2) F(41)2570(8) 1475(4) 1965(2) 139(2) C(122) 5045(7) 3962(4) 4744(2) 51(2)F(44) 5920(5) 3370(2) 4887(1) 74(1) F(45) 5616(5) 4563(2) 4959(1) 75(1)F(46) 3833(5) 3823(2) 4879(1) 75(1) C(108) 4748(13) 7054(5) 2773(3)100(3) F(47) 8547(9) 2143(4) 2026(2) 152(3) F(48) 6731(8) 1653(4)2180(2) 145(2) F(49) 10919(5) 3510(3) 4901(1) 81(1) C(123) 7913(9)2494(5) 2683(2) 73(2) C(121) 7997(12) 1902(6) 2341(3) 89(3) C(120)5377(8) −1834(4) 184(2) 53(2) C(109) 5564(11) −282(5) 2811(2) 79(3)F(55) 3970(4) −1704(2) 15(1) 70(1) F(54) 6048(5) −1235(2) 59(1) 67(1)F(56) 5748(5) −2425(2) −34(1) 69(1) F(53) 4868(7) −845(3) 2948(1) 100(2)F(51) 5179(13) 300(4) 3035(2) 189(4) F(52) 6924(8) −420(6) 2920(2)181(4) F(60) 8732(11) 1307(3) 2477(2) 192(4) *U(eq) is defined as onethird of the trace of the orthogonalized U^(ij) tensor.

In still yet an even further embodiment, the N-1 form of the compound ofExample 1 is substantially pure.

In still yet another embodiment, the N-1 form of the compound of Example1 contains at least about 90 wt. %, preferably at least about 95 wt. %,and more preferably at least about 99 wt. %, based on weight of the FormN-1 of the compound of Example 1.

In yet another embodiment, a substantially pure Form N-1 of the compoundof Example 1 has substantially pure phase homogeneity with less thanabout 10%, preferably less than about 5%, and more preferably less thanabout 2% of the total peak area of the experimentally measured PXRDpattern arising from peaks that are absent from the simulated PXRDpattern. Most preferably, the substantially pure crystalline Form N-1has substantially pure phase homogeneity with less than about 1% of thetotal peak area of the experimentally measured PXRD pattern arising frompeaks that are absent from the simulated PXRD pattern.

In another embodiment, the crystalline form of the compound of Example 1consists essentially of Form N-1. The crystalline form of thisembodiment may comprise at least about 90 wt. %, preferably at leastabout 95 wt. %, and more preferably at least about 99 wt. %, based onthe weight of the crystalline form, Form N-1 of the compound of Example1.

In yet another embodiment, a pharmaceutical composition is providedcomprising Form N-1 of the compound of Example 1; and at least onepharmaceutically-acceptable carrier and/or diluent.

In still another embodiment, a pharmaceutical composition comprisessubstantially pure Form N-1 of compound of Example 1; and at least onepharmaceutically-acceptable carrier and/or diluent.

In still an even further embodiment, a therapeutically effective amountof Form N-1 of the compound of Example 1 is combined with at least onepharmaceutically acceptable carrier and/or diluent to provide at leastone pharmaceutical composition.

In one embodiment, the compound of Example 1 is provided in a secondcrystalline form. The second crystalline form is an acetone solvatecrystalline form referred to herein as “Form A-2” or “A-2 Form”. The A-2Form comprises about one acetone molecule for each molecule of Example1.

In one embodiment, the A-2 Form is characterized by unit cell parametersapproximately equal to the following:

Cell dimensions:

-   -   a=9.25 Å    -   b=17.11 Å    -   c=19.63 Å    -   α=90.0°    -   β=99.2°    -   γ=90.0°

Space group: P2₁

Molecules of Example 1/asymmetric unit: 2

Volume/number of molecules in the unit cell=767 Å³

Density (calculated)=1.331 g/cm³,

wherein the unit cell parameters of Form A-2 are measured at atemperature of about −50° C.

In another embodiment, the A-2 Form is characterized by a simulatedpowder x-ray diffraction (PXRD) pattern substantially in accordance withthe pattern shown in FIG. 2 and/or by an observed PXRD patternsubstantially in accordance with the pattern shown in FIG. 2.

In yet an even further embodiment, the A-2 Form of Example 1 ischaracterized by fractional atomic coordinates substantially as listedin Table 2.

TABLE 2 Fractional Atomic Coordinates of Form A-2 of Example 1Calculated at aTemperature of about 25° C.; Atomic Coordinates (×10⁴)and Equivalent Isotropic Displacement Parameters (Å² × 10³) x y z U(eq)*C(7) 6904(4) 4997(2) 5292(2) 48(1) C(1) 8004(4) 5982(2) 6174(2) 54(1)C(2) 6800(4) 5545(2) 5859(2) 54(1) C(8) 6141(4) 4232(2) 5284(2) 54(1)C(3) 5457(5) 5651(3) 6082(2) 76(1) C(11) 4844(6) 2766(3) 5230(3) 85(2)C(6) 7848(6) 6490(3) 6720(2) 71(1) C(13) 6027(5) 3824(3) 5876(2) 70(1)C(9) 5582(5) 3890(3) 4653(2) 71(1) C(12) 5401(6) 3097(3) 5850(3) 83(2)C(10) 4917(6) 3170(3) 4628(3) 90(2) C(5) 6489(7) 6573(3) 6912(3) 90(2)C(4) 5305(7) 6149(4) 6600(3) 97(2) N(1) 9412(3) 5900(2) 5975(2) 51(1)N(2) 7595(3) 5151(2) 4788(2) 44(1) C(14) 10740(5) 5965(3) 6513(2) 74(1)O(2) 10864(3) 5937(2) 5145(1) 55(1) C(16) 8251(3) 5933(2) 4786(2) 40(1)C(15) 9646(4) 5938(2) 5311(2) 44(1) N(3) 8562(3) 6099(2) 4109(1) 41(1)C(17) 7467(4) 6272(2) 3606(2) 39(1) O(3) 6194(2) 6341(2) 3715(1) 51(1)C(18) 7818(4) 6368(2) 2879(2) 42(1) C(19) 7150(4) 5692(2) 2427(2) 43(1)C(20) 7742(4) 5710(2) 1743(2) 47(1) C(21) 7295(4) 7162(2) 2603(2) 53(1)C(22) 7920(6) 7834(3) 3079(2) 71(1) C(23) 7430(10) 8614(3) 2824(3)107(2) C(24) 7536(4) 4901(2) 2785(2) 52(1) C(25) 7093(6) 4206(3) 2330(2)71(1) C(26) 7241(8) 3455(3) 2704(3) 94(2) F(6) 6297(5) 3386(2) 3134(2)129(1) F(1) 7959(8) 9176(2) 3262(2) 185(3) F(5) 8543(6) 3362(2) 3105(3)150(2) F(3) 7942(5) 8794(2) 2229(2) 126(1) F(4) 7086(8) 2841(2) 2316(3)195(3) F(2) 5988(6) 8693(2) 2664(3) 144(2) O(4) 9043(3) 5579(2) 1739(1)64(1) N(4) 6801(3) 5840(2) 1174(2) 58(1) C(28) 3228(5) 5219(2) −666(2)58(1) C(27) 2067(5) 5688(2) −980(2) 58(1) C(34) 3784(6) 3921(3) −46(2)72(1) C(33) 3100(5) 4701(2) −78(2) 58(1) C(35) 4203(7) 3574(3) 594(3)97(2) C(39) 3986(6) 3507(3) −636(3) 83(2) C(32) 2285(6) 6154(3) −1536(2)75(1) C(30) 4731(7) 5711(3) −1475(3) 91(2) C(38) 4583(7) 2762(3) −576(3)98(2) C(29) 4568(6) 5253(3) −917(2) 72(1) C(31) 3577(7) 6155(3) −1784(3)87(2) C(37) 4968(9) 2422(4) 54(4) 118(2) C(36) 4771(9) 2808(4) 624(4)128(3) N(5) 661(4) 5680(2) −773(2) 60(1) N(6) 2443(4) 4911(2) 431(2)54(1) C(40) −651(6) 5774(3) −1302(2) 86(2) O(5) −740(3) 5772(2) 72(2)68(1) C(41) 469(4) 5735(2) −103(2) 53(1) C(42) 1863(4) 5705(2) 419(2)46(1) N(7) 1560(3) 5910(2) 1090(1) 45(1) C(43) 2620(4) 6162(2) 1576(2)46(1) O(6) 3897(3) 6255(2) 1483(1) 57(1) C(44) 2172(4) 6353(3) 2271(2)52(1) C(46) 2395(4) 5950(4) 3504(2) 74(1) C(45) 3085(4) 5884(3) 2850(2)62(1) C(48) 1358(6) 7707(3) 1848(3) 83(1) C(47) 2339(5) 7234(3) 2392(2)67(1) C(51) 1858(6) 4543(4) 2518(4) 107(2) C(50) 3255(4) 5013(3) 2687(2)70(1) C(49) 1563(10) 8561(4) 1908(5) 124(2) C(52) 2084(6) 3715(4)2468(4) 99(2) F(12) 3031(5) 3498(3) 2086(3) 142(2) F(10) 2663(6) 3415(3)3096(2) 163(2) F(9) 1342(6) 8818(3) 2530(3) 163(2) F(11) 888(5) 3295(3)2278(4) 183(3) F(8) 2877(7) 8787(3) 1859(4) 207(3) F(7) 614(7) 8955(3)1439(3) 173(2) O(7) 3291(3) 5999(2) 4102(2) 84(1) N(8) 1075(3) 5907(4)3480(2) 127(3) O(1S) 8859(5) 7601(2) 5406(3) 125(2) C(2S) 9451(6)8179(2) 5220(3) 93(2) C(1S) 8949(12) 8987(3) 5332(6) 178(4) C(3S)10720(8) 8060(6) 4852(5) 170(4) C(5S) 894(7) 8038(3) 9719(4) 114(2)C(6S) 1519(16) 8821(4) 9622(7) 237(7) C(4S) −672(9) 7980(10) 9801(11)366(14) O(2S) 1390(8) 7390(3) 9852(6) 226(4) *U(eq) is defined as onethird of the trace of the orthogonalized U^(ij) tensor.

In still yet an even further embodiment, the A-2 form of the compound ofExample 1 is substantially pure.

In still yet another embodiment, the A-2 form of the compound of Example1 contains at least about 90 wt. %, preferably at least about 95 wt. %,and more preferably at least about 99 wt. %, based on weight of thesecond crystalline form, Form A-2.

In yet another embodiment, a substantially pure second crystalline formhas substantially pure phase homogeneity with less than about 10%,preferably less than about 5%, and more preferably less than about 2% ofthe total peak area of the experimentally measured PXRD pattern arisingfrom peaks that are absent from the simulated PXRD pattern. Mostpreferably, a substantially pure second crystalline form hassubstantially pure phase homogeneity with less than about 1% of thetotal peak area of the experimentally measured PXRD pattern arising frompeaks that are absent from the simulated PXRD pattern.

In another embodiment, the second crystalline form of the compound ofExample 1 consists essentially of Form A-2. The second crystalline formof this embodiment may comprise at least about 90 wt. %, preferably atleast about 95 wt. %, and more preferably at least about 99 wt. %, basedon the weight of the second crystalline form, Form A-2.

In one embodiment, the compound of Example 1 is provided in a thirdcrystalline form. The third crystalline form is an ethyl acetate solvatecrystalline form referred to herein as “Form EA-3” or “EA-3 Form”. TheEA-3 Form comprises about one ethyl acetate molecule for each moleculeof Example 1.

In one embodiment, the EA-3 Form is characterized by unit cellparameters approximately equal to the following:

Cell dimensions:

-   -   a=8.84 Å    -   b=15.95 Å    -   c=22.38 Å    -   α=90.0°    -   β=90.0°    -   γ=90.0°

Space group: P2₁₂₁2₁

Molecules of Example 1/asymmetric unit: 1

Volume/number of molecules in the unit cell=789 Å³

Density (calculated)=1.357 g/cm³,

wherein the unit cell parameters of Form EA-3 are measured at atemperature of about −50° C.

In another embodiment, the EA-3 Form is characterized by a simulatedpowder x-ray diffraction (PXRD) pattern substantially in accordance withthe pattern shown in FIG. 3 and/or by an observed PXRD patternsubstantially in accordance with the pattern shown in FIG. 3.

In yet another embodiment, the EA-3 Form of the compound of Example 1 ischaracterized by a PXRD pattern (CuKα λ=1.5418 Å at a temperature ofabout 25° C.) comprising four or more, preferably five or more, 2θvalues selected from: 6.8±0.2, 9.6±0.2, 10.6±0.2, 15.4±0.2, 20.5±0.2,21.0±0.2, and 24.8±0.2, wherein the PXRD pattern of Form N-1 is measuredat a temperature of about 20° C.

In yet an even further embodiment, the EA-3 Form of Example 1 ischaracterized by fractional atomic coordinates substantially as listedin Table 3.

TABLE 3 Fractional Atomic Coordinates of Form EA-3 of Example 1Calculated at a Temperature of about 25° C.; Atomic Coordinates (×10⁴)and Equivalent Isotropic Displacement Parameters (Å² × 10³) x y z U(eq)*F(1) 10284(3) −324(1) 8889(1) 110(1) F(2) 8491(2) −284(1) 9520(1) 93(1)F(3) 10700(3) 44(1) 9784(1) 113(1) F(4) 10501(3) 5623(1) 9310(1) 130(1)F(5) 8241(3) 5391(1) 9141(1) 115(1) F(6) 8839(3) 5780(1) 9997(1) 105(1)O(1) 11040(2) 2804(1) 6756(1) 47(1) O(2) 7956(1) 2449(1) 8498(1) 36(1)O(3) 12740(2) 2982(1) 9716(1) 40(1) N(1) 8842(2) 2728(1) 6240(1) 33(1)N(2) 8089(2) 3668(1) 7331(1) 33(1) N(3) 9769(2) 2708(1) 7813(1) 33(1)N(4) 10970(2) 2584(1) 10384(1) 35(1) C(1) 7242(2) 2620(1) 6240(1) 31(1)C(2) 6305(2) 3136(1) 6588(1) 32(1) C(3) 4744(2) 2988(1) 6568(1) 41(1)C(4) 4146(3) 2361(2) 6218(1) 46(1) C(5) 5082(2) 1865(2) 5875(1) 45(1)C(6) 6619(2) 1993(2) 5884(1) 41(1) C(7) 6958(2) 3796(1) 6978(1) 32(1)C(8) 6335(2) 4667(1) 6961(1) 38(1) C(9) 6764(3) 5243(2) 7398(1) 49(1)C(10) 6266(3) 6066(2) 7374(1) 62(1) C(11) 5332(3) 6325(2) 6919(1) 65(1)C(12) 4902(3) 5765(2) 6489(1) 61(1) C(13) 5402(3) 4943(2) 6502(1) 49(1)C(14) 9661(2) 2771(1) 6748(1) 33(1) C(15) 8737(2) 2828(1) 7322(1) 31(1)C(16) 9646(3) 2733(1) 5664(1) 42(1) C(17) 9303(2) 2523(1) 8366(1) 28(1)C(18) 10560(2) 2404(1) 8823(1) 28(1) C(19) 10812(2) 1469(1) 8954(1)33(1) C(20) 9417(3) 1000(1) 9175(1) 40(1) C(21) 9715(3) 119(2) 9337(1)56(1) C(22) 10180(2) 2934(1) 9377(1) 28(1) C(23) 10038(2) 3867(1)9211(1) 35(1) C(24) 9480(3) 4403(1) 9721(1) 53(1) C(25) 9272(3) 5289(2)9554(1) 62(1) C(26) 11411(2) 2830(1) 9845(1) 28(1) O(1S) 3231(2) 3405(1)7982(1) 68(1) O(2S) 2969(2) 4783(1) 8077(1) 64(1) C(1S) 4926(3) 4166(2)8613(1) 78(1) C(2S) 3651(3) 4066(2) 8192(1) 54(1) C(3S) 1698(3) 4746(2)7661(1) 70(1) C(4S) 1132(4) 5596(2) 7579(2) 85(1) *U(eq) is defined asone third of the trace of the orthogonalized U^(ij) tensor.

In still yet an even further embodiment, the EA-3 form of the compoundof Example 1 is substantially pure.

In still yet another embodiment, the EA-3 form of the compound ofExample 1 contains at least about 90 wt. %, preferably at least about 95wt. %, and more preferably at least about 99 wt. %, based on weight ofthe third crystalline form, Form EA-3.

In yet another embodiment, a substantially pure Form EA-3 hassubstantially pure phase homogeneity with less than about 10%,preferably less than about 5%, and more preferably less than about 2% ofthe total peak area of the experimentally measured PXRD pattern arisingfrom peaks that are absent from the simulated PXRD pattern. Mostpreferably, the substantially crystalline Form EA-3 has substantiallypure phase homogeneity with less than about 1% of the total peak area ofthe experimentally measured PXRD pattern arising from peaks that areabsent from the simulated PXRD pattern.

In another embodiment, the third crystalline form of the compound ofExample 1 consists essentially of Form EA-3. The third crystalline formof this embodiment may comprise at least about 90 wt. %, preferably atleast about 95 wt. %, and more preferably at least about 99 wt. %, basedon the weight of the third crystalline form, Form EA-3.

In one embodiment, the compound of Example 1 is provided in a fourthcrystalline form. The fourth crystalline form is an tetrahydrofuransolvate crystalline form referred to herein as “Form THF-2” or “THF-2Form”. The THF-2 Form comprises about one tetrahydrofuran molecule foreach molecule of Example 1.

In one embodiment, the THF-2 Form is characterized by unit cellparameters approximately equal to the following:

Cell dimensions:

-   -   a=9.34 Å    -   b=16.44 Å    -   c=20.60 Å    -   α=90.0°    -   β=102.8°    -   γ=90.0°

Space group: P2₁

Molecules of Example 1/asymmetric unit: 2

Molecules of tetrahydrofuran/asymmetric unit: 2

Volume=3082 Å³,

wherein the unit cell parameters of Form THF-2 are measured at atemperature of about −50° C.

In another embodiment, the THF-2 Form is characterized by a simulatedpowder x-ray diffraction (PXRD) pattern substantially in accordance withthe pattern shown in FIG. 4 and/or by an observed PXRD patternsubstantially in accordance with the pattern shown in FIG. 4.

In yet another embodiment, the THF-2 Form of the compound of Example 1is characterized by a PXRD pattern (CuKα λ=1.5418 Å at a temperature ofabout 25° C.) comprising four or more, preferably five or more, 2θvalues selected from: 6.9±0.2, 9.6±0.2, 11.2±0.2, 12.6±0.2, 16.6±0.2,21.4±0.2, and 24.2±0.2, wherein the PXRD pattern of Form N-1 is measuredat a temperature of about 20° C.

In yet an even further embodiment, the THF-2 Form of Example 1 ischaracterized by fractional atomic coordinates substantially as listedin Table 4.

TABLE 4 Fractional Atomic Coordinates of Form THF-2 of Example 1 (NotIncluding Solvent Molecules) Calculated at a Temperature of about −50°C.; Atomic Coordinates (×10⁴) and Equivalent Isotropic DisplacementParameters (Å² × 10³) x y z U(eq)* C(15) 5518(8) 5421(5) −198(4) 40(2)C(7) 8183(7) 4416(4) −230(3) 37(2) C(1) 6912(8) 5409(5) −1086(3) 40(2)C(14) 7026(7) 5430(4) 285(3) 33(2) C(8) 8844(8) 3587(5) −216(4) 43(2)C(10) 9968(13) 2433(7) 425(5) 77(3) C(3) 9384(9) 5015(5) −1084(4) 53(2)C(13) 8784(8) 3143(5) −809(4) 48(2) C(9) 9434(11) 3222(6) 378(4) 63(2)C(2) 8146(8) 4952(5) −811(3) 39(2) C(16) 4171(9) 5392(7) −1345(4) 67(2)C(11) 9807(11) 1985(6) −145(5) 65(2) C(4) 9404(10) 5497(6) −1633(4)57(2) C(12) 9264(9) 2348(5) −782(4) 53(2) C(6) 6924(11) 5907(5) −1633(4)54(2) C(5) 8182(12) 5952(6) −1902(4) 64(3) N(1) 5578(6) 5360(4) −848(3)42(1) N(3) 6888(6) 5605(4) 938(3) 36(1) N(2) 7672(6) 4610(4) 275(3)35(1) O(1) 4390(5) 5416(4) −2(3) 52(1) O(2) 9346(5) 5736(3) 1309(2)39(1) C(18) 7891(7) 5843(4) 2128(3) 34(2) C(19) 8603(6) 5146(4) 2580(3)32(2) C(17) 8107(7) 5722(4) 1427(3) 31(2) C(24) 8791(9) 3596(5) 2706(4)48(2) C(23) 8173(8) 4312(5) 2258(3) 39(2) C(22) 8487(11) 8173(5) 2261(5)62(2) C(20) 8557(8) 6655(5) 2400(3) 39(2) C(21) 7907(9) 7385(5) 1964(4)49(2) C(25) 8741(10) 2810(5) 2355(4) 52(2) C(26) 8152(8) 5186(4) 3247(3)37(2) F(1) 9955(6) 8219(4) 2328(4) 93(2) F(2) 7946(8) 8805(4) 1891(4)99(2) F(3) 8223(9) 8310(4) 2860(3) 95(2) F(6) 9312(8) 2189(3) 2738(3)85(2) F(4) 9544(10) 2827(4) 1887(3) 107(3) F(5) 7371(8) 2601(4) 2055(4)99(2) O(3) 6868(5) 5095(4) 3269(2) 53(2) N(4) 9201(7) 5323(5) 3773(3)50(2) C(41) 5606(8) 5376(5) 5015(4) 44(2) C(33) 2941(8) 4383(5) 5034(4)44(2) C(40) 4123(8) 5383(5) 4524(3) 40(2) C(34) 2252(8) 3558(5) 5001(3)43(2) C(35) 1639(9) 3211(5) 4394(4) 50(2) C(28) 2933(9) 4914(5) 5586(4)46(2) C(39) 2268(11) 3105(6) 5556(4) 64(2) C(36) 1122(10) 2419(6)4377(4) 63(2) C(27) 4177(9) 5378(5) 5892(4) 49(2) C(29) 1675(10) 4985(6)5845(4) 57(2) C(30) 1676(13) 5507(7) 6397(5) 73(3) C(38) 1829(14)2307(7) 5543(5) 87(3) C(32) 4177(12) 5857(6) 6447(4) 62(2) C(37)1212(13) 1993(6) 4941(5) 78(3) C(31) 2903(15) 5910(7) 6692(5) 82(3) N(7)4371(6) 5572(4) 3879(3) 39(1) N(5) 3496(6) 4583(4) 4522(3) 40(1) N(6)5540(8) 5326(4) 5672(3) 52(2) C(42) 6922(10) 5333(7) 6176(4) 70(3) O(4)6769(6) 5366(4) 4854(3) 61(2) O(5) 1990(5) 5881(3) 3468(2) 47(1) C(43)3254(7) 5799(5) 3385(3) 37(2) C(44) 3604(7) 5919(5) 2715(3) 38(2) C(48)2633(7) 5381(5) 2189(3) 39(2) C(45) 3372(8) 6832(5) 2508(3) 42(2) C(47)4193(9) 8255(6) 2732(6) 68(3) C(49) 2530(8) 4500(5) 2392(4) 47(2) C(46)4402(9) 7411(5) 2964(4) 48(2) C(50) 3935(11) 4011(6) 2526(5) 64(2) F(7)5101(7) 8786(4) 3126(4) 96(2) F(8) 2886(6) 8555(4) 2680(4) 93(2) F(9)4504(7) 8366(4) 2111(3) 82(2) C(51) 3205(7) 5393(5) 1551(3) 43(2) N(8)2237(6) 5343(5) 980(3) 50(2) O(6) 4529(6) 5406(6) 1582(3) 84(2) C(52)3758(13) 3148(6) 2623(6) 75(3) F(12) 4965(9) 2712(5) 2742(5) 121(3)F(11) 2916(13) 2808(5) 2068(5) 146(4) F(10) 3056(12) 2973(5) 3096(5)148(4) *U(eq) is defined as one third of the trace of the orthogonalizedU^(ij) tensor.

In still yet an even further embodiment, the THF-2 form of the compoundof Example 1 is substantially pure.

In still yet another embodiment, the THF-2 form of the compound ofExample 1 contains at least about 90 wt. %, preferably at least about 95wt. %, and more preferably at least about 99 wt. %, based on weight ofthe fourth crystalline form, Form THF-2.

In yet another embodiment, a substantially pure Form THF-2 hassubstantially pure phase homogeneity with less than about 10%,preferably less than about 5%, and more preferably less than about 2% ofthe total peak area of the experimentally measured PXRD pattern arisingfrom peaks that are absent from the simulated PXRD pattern. Mostpreferably, the substantially crystalline Form THF-2 has substantiallypure phase homogeneity with less than about 1% of the total peak area ofthe experimentally measured PXRD pattern arising from peaks that areabsent from the simulated PXRD pattern.

In another embodiment, the fourth crystalline form of the compound ofExample 1 consists essentially of Form THF-2. The fourth crystallineform of this embodiment may comprise at least about 90 wt. %, preferablyat least about 95 wt. %, and more preferably at least about 99 wt. %,based on the weight of the fourth crystalline form, Form THF-2.

Crystalline Form of the Compound of Example 2

In one embodiment, the compound of Example 2

is provided as a crystalline material comprising a crystalline form. Anexample of a suitable crystalline form of the compound of Example 2 isForm M2-1. The M2-1 Form comprises about two methanol molecules for eachmolecule of Example 2.

In one embodiment, the M2-1 Form of compound of Example 2 ischaracterized by unit cell parameters approximately equal to thefollowing:

Cell dimensions:

-   -   a=8.44 Å    -   b=21.02 Å    -   c=17.52 Å    -   α=90.0°

β=90.88°

-   -   γ=90.0°

Space group: P2₁

Molecules of Example 2/asymmetric unit: 2

Volume/Number of molecules in the unit cell=777 Å³

Density (calculated)=1.297 g/cm³,

wherein the unit cell parameters of Form M-1 are measured at atemperature of about −100° C.

In another embodiment, the M2-1 Form is characterized by a simulatedpowder x-ray diffraction (PXRD) pattern substantially in accordance withthe pattern shown in FIG. 5 and/or by an observed PXRD patternsubstantially in accordance with the pattern shown in FIG. 5.

In yet another embodiment, the M2-1 Form of the compound of Example 2 ischaracterized by a PXRD pattern (CuKα λ=1.5418 Å at a temperature ofabout 25° C.) comprising four or more, preferably five or more, 2θvalues selected from: 8.2±0.2, 12.2±0.2, 14.2±0.2, 15.1±0.2, 16.8±0.2,17.3±0.2, and 23.0±0.2, wherein the PXRD pattern of Form M2-1 ismeasured at a temperature of about 20° C.

In yet an even further embodiment, the M2-1 Form of Example 2 ischaracterized by fractional atomic coordinates substantially as listedin Table 5.

TABLE 5 Fractional Atomic Coordinates of Form M2-1 Calculated at aTemperature of about 25° C.; Atomic Coordinates (×10⁴) and EquivalentIsotropic Displacement Parameters (Å² × 10³) for Example 2, Form M2-1 xy z U(eq) F(1) 2613(2) −212(1) 2456(1) 77(1) F(2) 4541(3) −625(1)3070(2) 96(1) F(3) 4934(3) −161(1) 2004(1) 96(1) F(4) 7167(3) 3758(1)3848(1) 77(1) F(5) 6914(3) 3210(1) 4862(1) 90(1) F(6) 9215(3) 3403(1)4441(1) 85(1) F(7) 7591(2) 3726(1) 11734(2) 119(1) F(8) 9635(3) 4107(1)11204(1) 86(1) F(9) 9848(3) 3673(1) 12297(1) 79(1) F(10) 12277(3)−275(1) 10384(1) 91(1) F(11) 11834(6) 315(1) 9441(2) 176(2) F(12)14222(4) 122(1) 9816(2) 133(1) O(1) 1635(2) 931(1) 4421(1) 37(1) O(2)5968(2) 825(1) 4908(1) 47(1) O(3) 8139(2) 1682(1) 2398(1) 35(1) O(4)6559(2) 2497(1) 9963(1) 39(1) O(5) 10872(2) 2663(1) 9445(1) 39(1) O(6)13114(2) 1784(1) 11956(1) 34(1) N(1) 2751(2) 2232(1) 5319(1) 34(1) N(2)1067(2) 1032(1) 5670(1) 33(1) N(3) 4363(2) 1622(1) 4536(1) 30(1) N(4)10420(2) 1368(1) 2964(1) 37(1) N(5) 7664(2) 1284(1) 8920(1) 33(1) N(6)5929(2) 2493(1) 8710(1) 31(1) N(7) 9312(2) 1845(1) 9778(1) 31(1) N(8)15396(2) 2076(1) 11411(1) 40(1) C(1) 1345(3) 1256(1) 6422(1) 35(1) C(2)1849(3) 1874(1) 6578(1) 36(1) C(3) 2048(3) 2059(2) 7348(2) 45(1) C(4)1720(3) 1644(2) 7931(2) 55(1) C(5) 1161(3) 1038(2) 7766(2) 56(1) C(6)981(3) 842(2) 7018(2) 44(1) C(7) 2098(3) 2347(1) 5969(1) 35(1) C(8)1531(3) 3012(1) 6086(2) 40(1) C(9) 2312(4) 3514(2) 5740(2) 51(1) C(10)1764(5) 4131(2) 5823(2) 65(1) C(11) 428(5) 4245(2) 6243(3) 80(1) C(12)−352(5) 3754(2) 6580(3) 80(1) C(13) 191(4) 3139(2) 6513(2) 56(1) C(14)1961(3) 1152(1) 5056(1) 30(1) C(15) 3364(3) 1594(1) 5200(1) 31(1) C(16)5555(3) 1219(1) 4426(1) 30(1) C(17) 6342(3) 1256(1) 3652(1) 28(1) C(18)8095(3) 1450(1) 3748(1) 31(1) C(19) 8887(3) 1501(1) 2979(1) 29(1) C(20)6151(3) 613(1) 3254(2) 36(1) C(21) 4416(3) 482(1) 3060(2) 39(1) C(22)4138(4) −124(2) 2655(2) 56(1) C(23) 8335(3) 2081(1) 4176(1) 34(1) C(24)7670(3) 2664(1) 3763(2) 39(1) C(25) 7738(4) 3248(2) 4225(2) 50(1) C(26)6216(3) 2346(1) 7935(1) 29(1) C(27) 6745(3) 1743(1) 7708(1) 29(1) C(28)6963(3) 1642(1) 6922(1) 37(1) C(29) 6629(3) 2113(2) 6398(2) 46(1) C(30)6065(3) 2691(2) 6633(2) 46(1) C(31) 5856(3) 2812(1) 7399(2) 39(1) C(32)6994(3) 1217(1) 8261(1) 30(1) C(33) 6421(3) 571(1) 8051(2) 35(1) C(34)7176(4) 43(1) 8360(2) 49(1) C(35) 6628(4) −563(2) 8178(2) 62(1) C(36)5318(4) −640(2) 7705(2) 63(1) C(37) 4561(4) −123(2) 7405(2) 59(1) C(38)5104(3) 486(1) 7577(2) 45(1) C(39) 6856(3) 2328(1) 9310(1) 30(1) C(40)8266(3) 1907(1) 9119(1) 30(1) C(41) 10484(3) 2259(1) 9915(1) 30(1) C(42)11305(3) 2212(1) 10692(1) 29(1) C(43) 13053(3) 2027(1) 10608(1) 28(1)C(44) 13850(3) 1958(1) 11389(1) 27(1) C(45) 11115(3) 2854(1) 11089(1)33(1) C(46) 9380(3) 2999(2) 11248(2) 44(1) C(47) 9111(4) 3620(2)11616(2) 59(1) C(48) 13303(3) 1408(1) 10152(1) 37(1) C(49) 12699(4)817(1) 10535(2) 46(1) C(50) 12765(6) 249(2) 10046(2) 71(1) O(1S) 3445(3)3322(1) 8972(1) 65(1) O(2S) 3658(2) 2451(1) 3295(1) 43(1) O(3S) 8681(2)1043(1) 1039(1) 51(1) C(4S) 2039(4) 4719(2) 3916(2) 64(1) C(1S) 3853(6)3821(2) 9485(3) 123(2) C(2S) 2594(4) 2953(2) 3435(2) 59(1) C(3S) 7938(7)447(2) 954(3) 107(2) O(4S) 1606(03) 5269(1) 4324(2) 63(1) U(eq) isdefined as one third of the trace of the orthogonalized U^(ij) tensor.

In still yet an even further embodiment, the M2-1 form of the compoundof Example 2 is substantially pure.

In still yet another embodiment, the M2-1 form of the compound ofExample 2 contains at least about 90 wt. %, preferably at least about 95wt. %, and more preferably at least about 99 wt. %, based on weight ofthe crystalline form, Form M2-1.

In yet another embodiment, a substantially pure crystalline form of FormM2-1 has substantially pure phase homogeneity with less than about 10%,preferably less than about 5%, and more preferably less than about 2% ofthe total peak area of the experimentally measured PXRD pattern arisingfrom peaks that are absent from the simulated PXRD pattern. Mostpreferably, the substantially pure crystalline form of M2-1 hassubstantially pure phase homogeneity with less than about 1% of thetotal peak area of the experimentally measured PXRD pattern arising frompeaks that are absent from the simulated PXRD pattern.

In another embodiment, the crystalline form of the compound of Example 2consists essentially of Form M2-1. The crystalline form of thisembodiment may comprise at least about 90 wt. %, preferably at leastabout 95 wt. %, and more preferably at least about 99 wt. %, based onthe weight of the crystalline form, Form M2-1.

Compounds in accordance with Formula (I) can be administered by anymeans suitable for the condition to be treated, which can depend on theneed for site-specific treatment or quantity of Formula (I) compound tobe delivered.

Also embraced within this invention is a class of pharmaceuticalcompositions comprising the compound of Formula (I) or prodrug thereof;and one or more non-toxic, pharmaceutically-acceptable carriers and/ordiluents and/or adjuvants (collectively referred to herein as “carrier”materials) and, if desired, other active ingredients. The compounds ofFormula (I) may be administered by any suitable route, preferably in theform of a pharmaceutical composition adapted to such a route, and in adose effective for the treatment intended. The compounds andcompositions of the present invention may, for example, be administeredorally, mucosally, or parentally including intravascularly,intravenously, intraperitoneally, subcutaneously, intramuscularly, andintrasternally in dosage unit formulations containing conventionalpharmaceutically acceptable carriers, adjuvants, and vehicles. Forexample, the pharmaceutical carrier may contain a mixture of mannitol orlactose and microcrystalline cellulose. The mixture may containadditional components such as a lubricating agent, e.g., magnesiumstearate and a disintegrating agent such as crospovidone. The carriermixture may be filled into a gelatin capsule or compressed as a tablet.The pharmaceutical composition may be administered as an oral dosageform or an infusion, for example.

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, suspension, or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient. Forexample, the pharmaceutical composition may be provided as a tablet orcapsule comprising an amount of active ingredient in the range of fromabout 1 to 2000 mg, preferably from about 1 to 500 mg, and morepreferably from about 5 to 150 mg. A suitable daily dose for a human orother mammal may vary widely depending on the condition of the patientand other factors, but, can be determined using routine methods.

Any pharmaceutical composition contemplated herein can, for example, bedelivered orally via any acceptable and suitable oral preparations.Exemplary oral preparations, include, but are not limited to, forexample, tablets, troches, lozenges, aqueous and oily suspensions,dispersible powders or granules, emulsions, hard and soft capsules,syrups, and elixirs. Pharmaceutical compositions intended for oraladministration can be prepared according to any methods known in the artfor manufacturing pharmaceutical compositions intended for oraladministration. In order to provide pharmaceutically palatablepreparations, a pharmaceutical composition in accordance with theinvention can contain at least one agent selected from sweeteningagents, flavoring agents, coloring agents, demulcents, antioxidants, andpreserving agents.

A tablet can, for example, be prepared by admixing at least one compoundof Formula (I) with at least one non-toxic pharmaceutically acceptableexcipient suitable for the manufacture of tablets. Exemplary excipientsinclude, but are not limited to, for example, inert diluents, such as,for example, calcium carbonate, sodium carbonate, lactose, calciumphosphate, and sodium phosphate; granulating and disintegrating agents,such as, for example, microcrystalline cellulose, sodium croscarmellose,corn starch, and alginic acid; binding agents, such as, for example,starch, gelatin, polyvinyl-pyrrolidone, and acacia; and lubricatingagents, such as, for example, magnesium stearate, stearic acid, andtalc. Additionally, a tablet can either be uncoated, or coated by knowntechniques to either mask the bad taste of an unpleasant tasting drug,or delay disintegration and absorption of the active ingredient in thegastrointestinal tract thereby sustaining the effects of the activeingredient for a longer period. Exemplary water soluble taste maskingmaterials, include, but are not limited to, hydroxypropylmethylcelluloseand hydroxypropylcellulose. Exemplary time delay materials, include, butare not limited to, ethyl cellulose and cellulose acetate butyrate.

Hard gelatin capsules can, for example, be prepared by mixing at leastone compound of Formula (I) with at least one inert solid diluent, suchas, for example, calcium carbonate; calcium phosphate; and kaolin.

Soft gelatin capsules can, for example, be prepared by mixing at leastone compound of Formula (I) with at least one water soluble carrier,such as, for example, polyethylene glycol; and at least one oil medium,such as, for example, peanut oil, liquid paraffin, and olive oil.

An aqueous suspension can be prepared, for example, by admixing at leastone compound of Formula (I) with at least one excipient suitable for themanufacture of an aqueous suspension. Exemplary excipients suitable forthe manufacture of an aqueous suspension, include, but are not limitedto, for example, suspending agents, such as, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, alginic acid, polyvinylpyrrolidone, gum tragacanth, andgum acacia; dispersing or wetting agents, such as, for example, anaturally-occurring phosphatide, e.g., lecithin; condensation productsof alkylene oxide with fatty acids, such as, for example,polyoxyethylene stearate; condensation products of ethylene oxide withlong chain aliphatic alcohols, such as, for exampleheptadecaethylene-oxycetanol; condensation products of ethylene oxidewith partial esters derived from fatty acids and hexitol, such as, forexample, polyoxyethylene sorbitol monooleate; and condensation productsof ethylene oxide with partial esters derived from fatty acids andhexitol anhydrides, such as, for example, polyethylene sorbitanmonooleate. An aqueous suspension can also contain at least onepreservative, such as, for example, ethyl and n-propylp-hydroxybenzoate; at least one coloring agent; at least one flavoringagent; and/or at least one sweetening agent, including but not limitedto, for example, sucrose, saccharin, and aspartame.

Oily suspensions can, for example, be prepared by suspending at leastone compound of Formula (I) in either a vegetable oil, such as, forexample, arachis oil; olive oil; sesame oil; and coconut oil; or inmineral oil, such as, for example, liquid paraffin. An oily suspensioncan also contain at least one thickening agent, such as, for example,beeswax; hard paraffin; and cetyl alcohol. In order to provide apalatable oily suspension, at least one of the sweetening agents alreadydescribed hereinabove, and/or at least one flavoring agent can be addedto the oily suspension. An oily suspension can further contain at leastone preservative, including, but not limited to, for example, ananti-oxidant, such as, for example, butylated hydroxyanisol, andalpha-tocopherol.

Dispersible powders and granules can, for example, be prepared byadmixing at least one compound of Formula (I) with at least onedispersing and/or wetting agent; at least one suspending agent; and/orat least one preservative. Suitable dispersing agents, wetting agents,and suspending agents are as already described above. Exemplarypreservatives include, but are not limited to, for example,anti-oxidants, e.g., ascorbic acid. In addition, dispersible powders andgranules can also contain at least one excipient, including, but notlimited to, for example, sweetening agents; flavoring agents; andcoloring agents.

An emulsion of at least one compound of Formula (I) can, for example, beprepared as an oil-in-water emulsion. The oily phase of the emulsionscomprising compounds of Formula (I) may be constituted from knowningredients in a known manner. The oil phase can be provided by, but isnot limited to, for example, a vegetable oil, such as, for example,olive oil and arachis oil; a mineral oil, such as, for example, liquidparaffin; and mixtures thereof. While the phase may comprise merely anemulsifier, it may comprise a mixture of at least one emulsifier with afat or an oil or with both a fat and an oil. Suitable emulsifying agentsinclude, but are not limited to, for example, naturally-occurringphosphatides, e.g., soy bean lecithin; esters or partial esters derivedfrom fatty acids and hexitol anhydrides, such as, for example, sorbitanmonooleate; and condensation products of partial esters with ethyleneoxide, such as, for example, polyoxyethylene sorbitan monooleate.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make-up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations. Anemulsion can also contain a sweetening agent, a flavoring agent, apreservative, and/or an antioxidant. Emulsifiers and emulsionstabilizers suitable for use in the formulation of the present inventioninclude Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol,glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate aloneor with a wax, or other materials well known in the art.

The compounds of Formula (I) can, for example, also be deliveredintravenously, subcutaneously, and/or intramuscularly via anypharmaceutically acceptable and suitable injectable form. Exemplaryinjectable forms include, but are not limited to, for example, sterileaqueous solutions comprising acceptable vehicles and solvents, such as,for example, water, Ringer's solution, and isotonic sodium chloridesolution; sterile oil-in-water microemulsions; and aqueous or oleaginoussuspensions.

Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules using one or more of the carriers or diluents mentioned for usein the formulations for oral administration or by using other suitabledispersing or wetting agents and suspending agents. The compounds may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, tragacanth gum, and/or various buffers. Other adjuvants andmodes of administration are well and widely known in the pharmaceuticalart. The active ingredient may also be administered by injection as acomposition with suitable carriers including saline, dextrose, or water,or with cyclodextrin (i.e., CAPTISOL®), cosolvent solubilization (i.e.,propylene glycol) or micellar solubilization (i.e., Tween 80).

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employed,including synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

A sterile injectable oil-in-water microemulsion can, for example, beprepared by 1) dissolving at least one compound of Formula (I) in anoily phase, such as, for example, a mixture of soybean oil and lecithin;2) combining the Formula (I) containing oil phase with a water andglycerol mixture; and 3) processing the combination to form amicroemulsion.

A sterile aqueous or oleaginous suspension can be prepared in accordancewith methods already known in the art. For example, a sterile aqueoussolution or suspension can be prepared with a non-toxicparenterally-acceptable diluent or solvent, such as, for example,1,3-butane diol; and a sterile oleaginous suspension can be preparedwith a sterile non-toxic acceptable solvent or suspending medium, suchas, for example, sterile fixed oils, e.g., synthetic mono- ordiglycerides; and fatty acids, such as, for example, oleic acid.

Pharmaceutically acceptable carriers, adjuvants, and vehicles that maybe used in the pharmaceutical compositions of this invention include,but are not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-alpha-tocopherol polyethyleneglycol 1000 succinate, surfactants usedin pharmaceutical dosage forms such as Tweens, polyethoxylated castoroil such as CREMOPHOR surfactant (BASF), or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as alpha-, beta-, and gamma-cyclodextrin,or chemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutically active compounds of this invention can be processedin accordance with conventional methods of pharmacy to produce medicinalagents for administration to patients, including humans and othermammals. The pharmaceutical compositions may be subjected toconventional pharmaceutical operations such as sterilization and/or maycontain conventional adjuvants, such as preservatives, stabilizers,wetting agents, emulsifiers, buffers etc. Tablets and pills canadditionally be prepared with enteric coatings. Such compositions mayalso comprise adjuvants, such as wetting, sweetening, flavoring, andperfuming agents.

The amounts of compounds that are administered and the dosage regimenfor treating a disease condition with the compounds and/or compositionsof this invention depends on a variety of factors, including the age,weight, sex, the medical condition of the subject, the type of disease,the severity of the disease, the route and frequency of administration,and the particular compound employed. Thus, the dosage regimen may varywidely, but can be determined routinely using standard methods. A dailydose of about 0.001 to 100 mg/kg body weight, preferably between about0.005 and about 50 mg/kg body weight and most preferably between about0.01 to 10 mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day.

For therapeutic purposes, the active compounds of this invention areordinarily combined with one or more adjuvants appropriate to theindicated route of administration. If administered orally, the compoundsmay be admixed with lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets maycontain a controlled-release formulation as may be provided in adispersion of active compound in hydroxypropylmethyl cellulose.

Pharmaceutical compositions of this invention comprise the compound ofFormula (I), or a prodrug thereof, and optionally an additional agentselected from any pharmaceutically acceptable carrier, adjuvant, andvehicle. Alternate compositions of this invention comprise a compound ofthe Formula (I) described herein, or a prodrug thereof, and apharmaceutically acceptable carrier, adjuvant, or vehicle.

Utility

The compounds of Formula (I) are useful for the treatment of cancer, forexample, cancers dependent upon Notch activation. Notch activation hasbeen implicated in the pathogenesis of various solid tumors includingovarian, pancreatic, as well as breast cancer and hematologic tumorssuch as leukemias, lymphomas, and multiple myeloma.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof. The method of this embodiment can be used to treat avariety of cancers, including, but not limited to, bladder cancer,breast cancer, colorectal cancer, gastric cancer, head and neck cancer,kidney cancer, liver cancer, lung cancer including non-small cell lungcancer (NSCLC), ovarian cancer, pancreatic cancer, gall bladder cancer,prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma,malignant fibrous histiocytoma (MFH), fibrosarcoma,glioblastomas/astrocytomas, neuroblastoma, melanoma, T-cell acutelymphoblastic leukemia (T-ALL), and mesothelioma. For example, themethod of this embodiment is used to treat breast cancer, colon cancer,or pancreatic cancer. Preferably, the mammal is a human. For example, atherapeutically effective amount for treating cancer may be administeredin the method of the present embodiment. The method of this embodimentincludes the administration of the compound having the structure:

The method of this embodiment also includes the administration of thecompound having the structure:

Routes of administration in the present embodiment include parenteraladministration and oral administration.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof, wherein said cancer is colorectal cancer. Preferably,the mammal is a human. For example, a therapeutically effective amountfor treating cancer may be administered in the method of the presentembodiment. Routes of administration in the present embodiment includeparenteral administration and oral administration.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof, wherein said cancer is triple negative breast cancer.Preferably, the mammal is a human. For example, a therapeuticallyeffective amount for treating cancer may be administered in the methodof the present embodiment. Routes of administration in the presentembodiment include parenteral administration and oral administration.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof, wherein said cancer is non-small cell lung cancer.Preferably, the mammal is a human. For example, a therapeuticallyeffective amount for treating cancer may be administered in the methodof the present embodiment. Routes of administration in the presentembodiment include parenteral administration and oral administration.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof, wherein said cancer is pancreatic cancer. Preferably,the mammal is a human. For example, a therapeutically effective amountfor treating cancer may be administered in the method of the presentembodiment. Routes of administration in the present embodiment includeparenteral administration and oral administration.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof, wherein said cancer is ovarian cancer. Preferably, themammal is a human. For example, a therapeutically effective amount fortreating cancer may be administered in the method of the presentembodiment. Routes of administration in the present embodiment includeparenteral administration and oral administration.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof, wherein said cancer is melanoma. Preferably, the mammalis a human. For example, a therapeutically effective amount for treatingcancer may be administered in the method of the present embodiment.Routes of administration in the present embodiment include parenteraladministration and oral administration.

In one embodiment, the use of a compound of Formula (I) or a prodrugthereof, in the manufacture of a medicament for the treatment of canceris provided. Preferably, in the present embodiment, cancers subject totreatment include one or more of bladder cancer, breast cancer,colorectal cancer, gastric cancer, head and neck cancer, kidney cancer,liver cancer, lung cancer including non-small cell lung cancer (NSCLC),ovarian cancer, pancreatic cancer, gall bladder cancer, prostate cancer,thyroid cancer, osteosarcoma, rhabdomyosarcoma, malignant fibroushistiocytoma (MFH), fibrosarcoma, glioblastomas/astrocytomas,neuroblastoma, melanoma, T-cell acute lymphoblastic leukemia (T-ALL),and mesothelioma. Suitable medicaments of the present embodiment includemedicaments for parenteral administration, such as, for example,solutions and suspensions and medicaments for oral administration, suchas, for example, tablets, capsules, solutions, and suspensions.

One embodiment provides a compound of Formula (I) or a prodrug thereof,for use in therapy in treating cancer. In the present embodiment,cancers subject to treatment include one or more of bladder cancer,breast cancer, colorectal cancer, gastric cancer, head and neck cancer,kidney cancer, liver cancer, lung cancer including non-small cell lungcancer (NSCLC), ovarian cancer, pancreatic cancer, gall bladder cancer,prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma,malignant fibrous histiocytoma (MFH), fibrosarcoma,glioblastomas/astrocytomas, neuroblastoma, melanoma, T-cell acutelymphoblastic leukemia (T-ALL), and mesothelioma.

In one embodiment, a method is provided for treating cancer in a mammalwherein the cancer is dependent upon Notch activation, comprisingadministering to the patient a compound of Formula (I) or a prodrugthereof. The method of this embodiment can be used to treat a variety ofcancers, including, but not limited to, bladder cancer, breast cancer,colorectal cancer, gastric cancer, head and neck cancer, kidney cancer,liver cancer, lung cancer including non-small cell lung cancer (NSCLC),ovarian cancer, pancreatic cancer, gall bladder cancer, prostate cancer,thyroid cancer, osteosarcoma, rhabdomyosarcoma, malignant fibroushistiocytoma (MFH), fibrosarcoma, glioblastomas/astrocytomas,neuroblastoma, melanoma, T-cell acute lymphoblastic leukemia (T-ALL),and mesothelioma. Preferably, the method of this embodiment is used totreat breast cancer, colon cancer, or pancreatic cancer. Preferably, themammal is a human. For example, a therapeutically effective amount fortreating cancer may be administered in the method of the presentembodiment. Suitable routes of administration include parenteraladministration and oral administration.

In treating cancer, a combination of chemotherapeutic agents and/orother treatments (e.g., radiation therapy) is often advantageous. Thesecond (or third) agent may have the same or different mechanism ofaction than the primary therapeutic agent. For example, drugcombinations may be employed wherein the two or more drugs beingadministered act in different manners or in different phases of the cellcycle, and/or where the two or more drugs have nonoverlapping toxicitiesor side effects, and/or where the drugs being combined each has ademonstrated efficacy in treating the particular disease statemanifested by the patient.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof; and administering one or more additional anti-canceragents.

The phrase “additional anti-cancer agent” refers to a drug selected fromany one or more of the following: alkylating agents (including nitrogenmustards, alkyl sulfonates, nitrosoureas, ethylenimine derivatives, andtriazenes); anti-angiogenics (including matrix metalloproteinaseinhibitors); antimetabolites (including adenosine deaminase inhibitors,folic acid antagonists, purine analogues, and pyrimidine analogues);antibiotics or antibodies (including monoclonal antibodies, CTLA-4antibodies, anthracyclines); aromatase inhibitors; cell-cycle responsemodifiers; enzymes; farnesyl-protein transferase inhibitors; hormonaland antihormonal agents and steroids (including synthetic analogs,glucocorticoids, estrogens/anti-estrogens [e.g., SERMs],androgens/anti-androgens, progestins, progesterone receptor agonists,and luteinizing hormone-releasing [LHRH] agonists and antagonists);insulin-like growth factor (IGF)/insulin-like growth factor receptor(IGFR) system modulators (including IGFR1 inhibitors);integrin-signaling inhibitors; kinase inhibitors (including multi-kinaseinhibitors and/or inhibitors of Src kinase or Src/abl, cyclin dependentkinase [CDK] inhibitors, panHer, Her-1 and Her-2 antibodies, VEGFinhibitors, including anti-VEGF antibodies, EGFR inhibitors,mitogen-activated protein [MAP] inhibitors, MET inhibitors, MEKinhibitors, Aurora kinase inhibitors, PDGF inhibitors, and othertyrosine kinase inhibitors or serine/threonine kinase inhibitors;microtubule-disruptor agents, such as ecteinascidins or their analogsand derivatives; microtubule-stabilizing agents such as taxanes, and thenaturally-occurring epothilones and their synthetic and semi-syntheticanalogs; microtubule-binding, destabilizing agents (including vincaalkaloids); topoisomerase inhibitors; prenyl-protein transferaseinhibitors; platinum coordination complexes; signal transductioninhibitors; and other agents used as anti-cancer and cytotoxic agentssuch as biological response modifiers, growth factors, and immunemodulators.

Accordingly, the compounds of the present invention may be administeredin combination with other anti-cancer treatments useful in the treatmentof cancer or other proliferative diseases. The invention herein furthercomprises use of a compound of Formula (I) or prodrug thereof inpreparing medicaments for the treatment of cancer, and/or it comprisesthe packaging of a compound of Formula (I) herein together withinstructions that the compound be used in combination with otheranti-cancer or cytotoxic agents and treatments for the treatment ofcancer. The present invention further comprises combinations of acompound of Formula (I) and one or more additional agents in kit form,e.g., where they are packaged together or placed in separate packages tobe sold together as a kit, or where they are packaged to be formulatedtogether.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof; administering dasatinib; and optionally, one or moreadditional anti-cancer agents.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof; administering paclitaxel; and optionally, one or moreadditional anti-cancer agents.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof; administering Tamoxifen; and optionally, one or moreadditional anti-cancer agents.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof; administering a glucocorticoid; and optionally, one ormore additional anti-cancer agents. An example of a suitableglucocorticoid is dexamethasone.

In one embodiment, a method is provided for treating cancer comprisingadministering to a mammal in need thereof a compound of Formula (I) or aprodrug thereof; administering carboplatin; and optionally, one or moreadditional anti-cancer agents.

The compounds of the present invention can be formulated orco-administered with other therapeutic agents that are selected fortheir particular usefulness in addressing side effects associated withthe aforementioned conditions. For example, compounds of the inventionmay be formulated with agents to prevent nausea, hypersensitivity andgastric irritation, such as antiemetics, and H₁ and H₂ antihistaminics.

In one embodiment, pharmaceutical compositions are provided comprising acompound of Formula (I) or prodrug thereof; one or more additionalagents selected from a kinase inhibitory agent (small molecule,polypeptide, and antibody), an immunosuppressant, an anti-cancer agent,an anti-viral agent, anti-inflammatory agent, antifungal agent,antibiotic, or an anti-vascular hyperproliferation compound; and anypharmaceutically acceptable carrier, adjuvant or vehicle.

The above other therapeutic agents, when employed in combination withthe compounds of the present invention, may be used, for example, inthose amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art. In the methodsof the present invention, such other therapeutic agent(s) may beadministered prior to, simultaneously with, or following theadministration of the inventive compounds.

The specific dose level and frequency of dosage for any particularsubject however, may be varied and generally depends on a variety offactors, including, but not limited to, for example, the bioavailabilityof the specific compound of Formula (I) in the administered form,metabolic stability and length of action of the specific compound ofFormula (I), species, body weight, general health, sex, diet of subject,mode and time of administration, rate of excretion, drug combination,and severity of the particular condition. For example, a daily dose ofabout 0.001 to 100 mg/kg body weight, preferably between about 0.005 andabout 50 mg/kg body weight and most preferably between about 0.01 to 10mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day.

The administration can be continuous, i.e., every day, orintermittently. The terms “intermittent” or “intermittently” as usedherein mean stopping and starting at either regular or irregularintervals. For example, intermittent administration includesadministration one to six days per week; administration in cycles (e.g.,daily administration for two to eight consecutive weeks followed by arest period with no administration for up to one week); oradministration on alternate days.

In one embodiment, the compound of Formula (I) is administeredcontinuously to a patient in need thereof, one or more times daily. Forexample, a therapeutically effective amount of the compound of Formula(I) is administered to a patient in need thereof, one or more timesdaily for continuous days.

In one embodiment, the compound of Formula (I) is administeredintermittently to a patient in need thereof, one or more times daily.For example, a therapeutically effective amount of the compound ofFormula (I) is administered to a patient in need thereof, one or moretimes daily according to an intermittent schedule.

In one embodiment, the compound of Formula (I) is administered to apatient in need thereof, one or more times daily for continuous daysfollowed by one or more days without administration. Preferably, atherapeutically effective amount of the compound of Formula (I) isadministered. Examples of continuous dosing with a drug holiday arecycles of: 7 days on treatment followed by 7 days off treatment; 14 dayson treatment followed by 7 days off treatment; and 7 days on treatmentfollowed by 14 days off treatment. A cycle of on treatment/off treatmentcan be repeated multiple times as required to treat a patient.

In one embodiment, the compound of Formula (I) is administered to apatient in need thereof, according to an intermittent dosing schedule.Intermittent dosing schedules are repeating schedules including days inwhich the patient is administered the compound of Formula (I) and daysin which the patient is not administered the compound of Formula (I).Examples of intermittent dosing schedules are: dosing four days eachweek for three continuous weeks followed by a week without dosing, andrepeating on a four week interval; dosing five days each week for twocontinuous weeks followed by a week without dosing, and repeating on athree week interval; and dosing four days each week for one weekfollowed by two weeks without dosing, and repeating on a three weekinterval. Preferably, a therapeutically effective amount of the compoundof Formula (I) is administered.

In one embodiment, the compound of Formula (I) is administered on oneday, followed by 6 days of rest, and repeated on a weekly schedule.

In one embodiment, the compound of Formula (I) is administered on twoconsecutive days, followed by 5 days of rest, and repeated on a weeklyschedule.

In one embodiment, the compound of Formula (I) is administered on threeconsecutive days followed by four days of rest, and repeated on a weeklyschedule.

In one embodiment, the compound of Formula (I) is administered on oneday, followed by 10 to 13 days of rest.

Methods of Preparation

The compounds of the present invention can be prepared in a number ofways well known to one skilled in the art of organic synthesis. Thecompounds of the present invention can be synthesized using the methodsdescribed below, together with synthetic methods known in the art ofsynthetic organic chemistry, or variations thereon as appreciated bythose skilled in the art. Preferred methods include, but are not limitedto, those described below. All references cited herein are herebyincorporated in their entirety by reference.

The compounds of this invention may be prepared using the reactions andtechniques described in this section. The reactions are performed insolvents appropriate to the reagents and materials employed and aresuitable for the transformations being effected. Also, in thedescription of the synthetic methods described below, it is to beunderstood that all proposed reaction conditions, including choice ofsolvent, reaction atmosphere, reaction temperature, duration of theexperiment and work up procedures, are chosen to be the conditionsstandard for that reaction, which should be readily recognized by oneskilled in the art. It is understood by one skilled in the art oforganic synthesis that the functionality present on various portions ofthe molecule must be compatible with the reagents and reactionsproposed. Such restrictions to the substituents that are compatible withthe reaction conditions will be readily apparent to one skilled in theart and alternate methods must then be used. This will sometimes requirea judgment to modify the order of the synthetic steps or to select oneparticular process scheme over another in order to obtain a desiredcompound of the invention. It will also be recognized that another majorconsideration in the planning of any synthetic route in this field isthe judicious choice of the protecting group used for protection of thereactive functional groups present in the compounds described in thisinvention. An authoritative account describing the many alternatives tothe trained practitioner is Greene et al. (Protective Groups in OrganicSynthesis, Third Edition, Wiley and Sons (1999)).

Compounds of Formula (I) may be prepared by reference to the methodsillustrated in the following Schemes. As shown therein the end productis a compound having the same structural formula as Formula (I). It willbe understood that any compound of Formula (I) may be produced by theschemes by the suitable selection of reagents with appropriatesubstitution. Solvents, temperatures, pressures, and other reactionconditions may readily be selected by one of ordinary skill in the art.Starting materials are commercially available or readily prepared by oneof ordinary skill in the art. Constituents of compounds are as definedherein or elsewhere in the specification.

The synthesis of the compounds of Formula (I) can be made using themethods summarized in Schemes 1 to 5.

The preparation of benzodiazepinone (iv) may be accomplished inmultitude of methods known to one skilled in the art. For example, asshown in Scheme 1, an appropriately substituted 2-aminobenzophenone (i)(for example, from Walsh, D. A., Synthesis, 677 (1980); and referencescited therein, or other methods known to one skilled in the art) may becoupled to the protected glycine derivative (ii) (PG=protecting group,for example PG=CBz, see Katritzky, A. R., J. Org. Chem., 55:2206-2214(1990)), treated with a reagent such as ammonia and subjected tocyclization to afford the benzodiazepinone (iii), according to theprocedure outlined in the literature (for example Sherrill, R. G. etal., J. Org. Chem., 60:730 (1995); or other routes known to one skilledin the art). The resulting racemic mixture may be separated (usingprocedures known to one skilled in the art) to get the individualenantiomers, or used as a racemate. Also, if R₃=H, (iii) may be, forexample, treated with a reagent such as MeI and a base such as K₂CO₃ ina solvent such as DMF to prepare R₃=Me.

Step 2: The deprotection of (iii) may be accomplished in several waysknown to one skilled in the art. For example, with PG=CBz, Compound(iii) may be treated with a reagent such as HBr in a solvent such asAcOH. Compound (iv) may be used as a racemate. Alternatively, compound(iv) may be subjected to enantiomeric resolution using standard methods(e.g., chiral preparative chromatography).

Step 1: The first step of Scheme 2 is accomplished by convertingcompound (v) to the ester (vii), employing one of the multiple waysknown to one skilled in the art, such as treatment with a substitutedacetimidate such as compound (vi) in the presence of a reagent such asboron trifluoride etherate at an appropriate temperature in a solventsuch as THF.

Step 2: Acid (viii) can be converted to compound (ix) in multiple waysknown to one skilled in the art. For example, treatment of acid (viii)with a reagent such as oxalyl chloride in a solvent such as DCM givesthe acid chloride (ix). Compound (ix) can be treated with anoxazolidinone (x) under standard conditions to give compound (xi)(Evans, D. A. et al., J. Am. Chem Soc., 112:4011 (1990)).

Step 3: Compound (xi) can be converted to compound (xii) in multipleways (Baran, P. et al., J. Am. Chem. Soc., 130(34):11546 (2008)). Forexample, compound (vii) is treated with a base such as LDA in a solventsuch as toluene, at low temperature such as −78° C. under an inertatmosphere such as N₂. The resulting mixture is added to a solution ofcompound (xi) treated with lithium chloride and a base such as LDA in asolvent such as toluene under an inert atmosphere such as N₂. To theresulting mixture of the enolates of compounds (vii) and (xi) is added acompound, such as bis(2-ethylhexanoyloxy)copper, at a low temperaturesuch as −78° C. under an inert atmosphere such as N₂ and warmed to roomtemperature to provide compound (xii).

Step 4: Conversion of compound (xii) to (xiii) may be accomplished bytreating it with reagents such as hydrogen peroxide and lithiumhydroxide at an appropriate temperature, using a mixture of solventssuch as THF/water. If necessary, the diastereoisomers may be separatedat this point via silica gel chromatography or preparative HPLC.Alternately, the mixture may be subjected to epimerization conditions,for example by treatment with LDA and diethylaluminum chloride followedby quenching with methanol or acetic acid to enrich the desireddiastereoisomer.

Step 5: Compound (xiii) may be coupled with benzodiazepinone (iv) in thepresence of a coupling reagent such as TBTU and a base such as TEA, in asolvent such as DMF to provide compound (xiv).

Step 6: Treatment of compound (xiv) with an acid such as TFA at anappropriate temperature such as 0° C., in a solvent such as DCM providescompound (xv).

Step 7: Conversion of compound (xv) to compound (xvi) may beaccomplished via coupling of compound (xv) with an appropriate aminesource such as ammonium chloride, a carbodiimide such as EDC, HOBT and abase such as TEA in a solvent such as DMF. If necessary thediastereoisomeric mixture can be separated using an appropriateseparation technique, such as chiral preparative chromatography.

Step 1: The preparation of benzodiazepinone (iii) may also beaccomplished by cross coupling of benzodiazepinone (xvii) containing ahalogen atom such as chlorine (X=Cl) and a protecting group (PG) such asBoc, with an appropriate coupling partner such as a boronic acid underconditions known to one skilled in the art. For example, the coupling ofthe halogen containing moiety with a boronic acid occurs in the presenceof a catalyst such as tetrakis(triphenylphosphine)palladium(0), a basesuch as sodium carbonate and a solvent such as DME under an inertatmosphere such as N₂.

Step 1: The first step of Scheme 4 involves the treatment of Compound(xvii) with carboxylic acid (xix) in the presence of a carbodiimide suchas DCC, a base such as TEA, and a catalyst such as DMAP in a solventsuch as DCM provides Compound (xx).

Step 2: Conversion of Compound (xx) to Compound (xxi) may beaccomplished by treatment with a reagent such as sodium cyanoborohydridein the presence of an acid such as HCl under atmospheric conditions thatmay be inert, for example under N₂.

Step 3: Conversion of Compound (xxi) to Compound (xxiii) may proceed via

Compound (xxii) bearing an appropriate leaving group (LG). For example,treatment of Compound (xxi) with a base such as 2,6-lutidine and areagent such as trifluoromethanesulfonic anhydride in a solvent such asDCM at an appropriate temperature such as −78° C., provides the triflateof Compound (xxii). Compound (xxii) may now be subjected to crosscoupling reaction conditions to provide Compound (xxiii). For example,treatment of Compound (xxii) with an appropriately substituted couplingpartner, for example a boronic acid, in the presence of a catalyst suchas tetrakis(triphenylphosphine)palladium(0), a base such as potassiumphosphate in a solvent such as dioxane under atmospheric conditions thatmay be inert, for example under N₂, provides Compound (xxiii).

Step 4: Conversion of Compound (xxiii) to Compound (xxiv) may beaccomplished via standard procedures known to one skilled in the art.For example, treatment of Compound (xxiii) in the presence of a catalystsuch as Pd/C in a solvent such as methanol gives Compound (xxiv).

Step 5: Compound (xxv) may be obtained by the coupling of Compound(xxiv) with Compound (iv). For example, the transformation may beaccomplished with the use of a reagent such as AlMe₃ in a solvent suchas DCM under an inert atmosphere such as N₂. At this instance themixture of diastereoisomers obtained may be used as a mixture or may beseparated by an appropriate method such as chiral chromatography.

Step 6: Compound (xxv) is oxidized using an oxidizing agent such asJones reagent, in a solvent such as acetone to give Compound (xxvi). Ifthe compound is a diastereoisomeric mixture then it may be used as amixture or may be separated using an appropriate method such as chiralchromatography.

Step 7: Conversion of Compound (xxvi) to Compound (xxvii) may beaccomplished via standard procedures known to one skilled in the art.For example, coupling of Compound (xxvi) with an appropriate aminesource such as ammonium chloride, a carbodiimide such as EDC, HOBT and abase such as TEA in a solvent such as DMF provides Compound (xxvii). Atthis instance the compound may be enantiopure or if necessary thediastereoisomeric mixture can be separated using an appropriateseparation technique, such as chiral chromatography.

Step 1: The first step of Scheme 5 is accomplished by treating Compound(xxviii) with a reagent such as sodium nitrite in an acid such as H₂SO₄and a solvent such as water to provide Compound xxix.

Step 2: The acid (xxix) is converted to compound (xxx) (PG=protectinggroup). For example, the acid (xxix) is treated with an alcohol such asbenzyl alcohol in a solvent such as toluene and an acid such as H₂SO₄ toprovide Compound xxx.

Step 3: Compound (xxxi) bearing a suitable leaving group may be preparedby treatment of Compound (xxx) with a base such as 2,6-lutidine and areagent such as trifluoromethanesulfonic anhydride in a solvent such asDCM at an appropriate temperature.

Step 4: Compound (xxxii) can be converted to Compound (xxxiv) inmultiple ways known to one skilled in the art. For example, treatment ofacid chloride (xxxii), either prepared from the corresponding carboxylicacid with a reagent such as oxalyl chloride in a solvent such as DCM, orobtained commercially, can be treated with an oxazolidinone (xxxiii)under standard conditions to give Compound (xxxiv) (Evans, D. A. et al.,J. Am. Chem. Soc., 112:4011 (1990)).

Step 5: The preparation of Compound (xxxv) may be effected by treatingCompound (xxxiv) with a base such as LiHMDS in a solvent such as THF atan appropriate temperature such as −78° C. and to the resulting mixtureis added Compound (xxxi) in a solvent such as THF.

Step 6: The protecting group of Compound (xxxv) may be removed via manymethods known to one skilled in the art. For example, a benzyl group maybe removed by subjecting it to hydrogenation conditions using apalladium catalyst such as Pearlman's Catalyst in a solvent such asmethanol to provide Compound (xxxvi).

Step 7: Compound (iv) is coupled with Compound (xxxvi) in the presenceof a coupling reagent such as TBTU and a base such as TEA in a solventsuch as DMF to provide Compound (xxxvii). If necessary, thediastereoisomers may be separated using an appropriate method such aschiral preparative chromatography.

Step 8: The hydrolysis of Compound (xxxvii) may be accomplished bytreating it with hydrogen peroxide and lithium hydroxide at anappropriate temperature using a mixture of solvents such as THF/water togive Compound (xv). If necessary, the diastereoisomers may be separatedusing an appropriate method such as chiral preparative chromatography.

Compound (xiii) in Scheme 2 may also be prepared from compound (xi) bysynthetic sequence outlined in Scheme 6.

Step 1: The first step of Scheme 6 is accomplished by treating Compound(xi) with a base such as sodium bis(trimethylsilyl)amide in a solventsuch as THF at low temperature such as −78° C. under an inertatmosphere. To the resulting enolate of (xi) is treated with a reagentsuch as tert-butyl bromoacetate to provide compound (xxxviii).

Step 2: Conversion of compound (xxxviii) to (xxxix) may be accomplishedby treating compound (xxxviii) with reagents such as hydrogen peroxideand lithium hydroxide at an appropriate temperature using a mixture ofsolvents such as THF/water.

Step 3: Compound (xxxix) can be converted to compound (xiii) bygenerating the enolate of (xxxix) with a base such as LDA in a solventsuch as THF at low temperature such as −78° C. under an inert atmosphereand further treatment with a reagent (R₂-LG) bearing an appropriateleaving group (e.g., LG=triflate).

EXAMPLES

The invention is further defined in the following Examples. It should beunderstood that the Examples are given by way of illustration only. Fromthe above discussion and the Examples, one skilled in the art canascertain the essential characteristics of the invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications to adapt the invention to various uses and conditions.As a result, the invention is not limited by the illustrative examplesset forth hereinbelow, but rather is defined by the claims appendedhereto.

Abbreviations

AcOH acetic acidACN acetonitrileAlMe₃ trimethyl aluminumBoc tert-butyloxycarbonylDCC 1,3-dicyclohexylcarbodiimideDCM dichloromethaneDEA diethylamineDMAP dimethylaminopyridineDME dimethyl etherDMF dimethylformamideDMSO dimethyl sulfoxideEDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlorideEDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimideEt₂AlCl diethyl aluminum chlorideEtOAc ethyl acetateH₂SO₄ sulfuric acidHCl hydrochloric acidHOBT hydroxybenzotriazole

HPLC High Performance Liquid Chromatography

hr hour(s)IPA isopropyl alcohol

LCMS Liquid Chromatography-Mass Spectroscopy

LDA lithium diisopropylamideLiHMDS lithium bis(trimethylsilyl)amideMe methylMeOH methanolmin minute(s)MTBE methyl tert-butyl etherN₂ nitrogenNaHMDS sodium bis(trimethylsilyl)amidePd/C palladium on carbonPh phenylRT retention timesat saturatedTBTU O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborateTEA triethylamineTf₂O trifluoromethylsulfonic anhydrideTFA trifluoroacetic acidTHF tetrahydrofuran

Example 1(2R,3S)—N-((3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide

Preparation 1A: tert-Butyl 5,5,5-trifluoropentanoate

To a stirred solution of 5,5,5-trifluoropentanoic acid (5 g, 32.0 mmol)in THF (30 mL) and hexane (30 mL) at 0° C., was added tert-butyl2,2,2-trichloroacetimidate (11.46 mL, 64.1 mmol). The mixture wasstirred for 15 min at 0° C. Boron trifluoride etherate (0.406 mL, 3.20mmol) was added and the reaction mixture was allowed to warm to roomtemperature overnight. To the clear reaction mixture was added solidNaHCO₃ (5 g) and stirred for 30 min. The mixture was filtered throughMgSO₄ and washed with hexanes (200 mL). The solution was allowed to restfor 45 min, and the resulting solid material was removed by filtering onthe same MgSO₄ filter again, washed with hexanes (100 mL) andconcentrated under reduced pressure without heat. The volume was reducedto about 30 mL, filtered through a clean fritted funnel, washed withhexane (5 mL), and then concentrated under reduced pressure withoutheat. The resulting neat oil was filtered through a 0.45 μm nylonmembrane filter disk to provide tert-butyl 5,5,5-trifluoropentanoate(6.6 g, 31.4 mmol 98% yield) as a colorless oil: ¹H NMR (400 MHz, CDCl₃)δ ppm 1.38 (s, 9H) 1.74-1.83 (m, 2H) 2.00-2.13 (m, 2H) 2.24 (t, J=7.28Hz, 2H).

Preparation 1B:(4S)-4-(Propan-2-yl)-3-(5,5,5-trifluoropentanoyl)-1,3-oxazolidin-2-one

To a stirred solution of 5,5,5-trifluoropentanoic acid (5.04 g, 32.3mmol) in DCM (50 mL) and DMF (3 drops) was added oxalyl chloride (3.4mL, 38.8 mmol) dropwise over 5 min and the solution was stirred untilall bubbling subsided. The reaction mixture was concentrated underreduced pressure to give pale yellow oil. To a separate flask chargedwith a solution of (4S)-4-(propan-2-yl)-1,3-oxazolidin-2-one (4.18 g,32.4 mmol) in THF (100 mL) at −78° C. was added n-BuLi (2.5M in hexane)(13.0 mL, 32.5 mmol) dropwise via syringe over 5 min. After stirring for10 min, the above acid chloride dissolved in THF (20 mL) was added viacannula over 15 min. The reaction mixture was warmed to 0° C., and wasallowed to warm to room temperature as the bath warmed and stirredovernight. To the reaction mixture was added saturated NH₄Cl, and thenextracted with EtOAc (2×). The combined organics were washed with brine,dried (Na₂SO₄), filtered and concentrated under reduced pressure. Thecrude material was purified by flash chromatography (Teledyne ISCOCombiFlash Rf, 5% to 60% solvent A/B=hexanes/EtOAc, REDISEP® SiO₂ 120g). Concentration of appropriate fractions provided Preparation 1B (7.39g, 86%) as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ ppm 4.44 (1H, dt,J=8.31, 3.53 Hz), 4.30 (1H, t, J=8.69 Hz), 4.23 (1H, dd, J=9.06, 3.02Hz), 2.98-3.08 (2H, m), 2.32-2.44 (1H, m, J=13.91, 7.02, 7.02, 4.03 Hz),2.13-2.25 (2H, m), 1.88-2.00 (2H, m), 0.93 (3H, d, J=7.05 Hz), 0.88 (3H,d, J=6.80 Hz).

Preparation 1C: (2S,3R)-tert-Butyl6,6,6-trifluoro-3-((S)-4-isopropyl-2-oxooxazolidine-3-carbonyl)-2-(3,3,3-trifluoropropyl)hexanoate,and Preparation 1D: (2R,3R)-tert-Butyl6,6,6-trifluoro-3-((S)-4-isopropyl-2-oxooxazolidine-3-carbonyl)-2-(3,3,3-trifluoropropyl)hexanoate

To a cold (−78° C.), stirred solution of diisopropylamine (5.3 mL, 37.2mmol) in THF (59 mL) under nitrogen atmosphere was added n-BuLi (2.5M inhexane) (14.7 mL, 36.8 mmol), then warmed to 0° C. to give a 0.5Msolution of LDA. A separate vessel was charged with Preparation 1B (2.45g, 9.17 mmol), the material was azeotroped twice with benzene (theRotoVap air inlet was fitted with nitrogen inlet to completely excludehumidity) then toluene (15.3 mL) was added. This solution was added to aflask containing dry lithium chloride (1.96 g, 46.2 mmol). To theresultant mixture, cooled to −78° C., was added LDA solution (21.0 mL,10.5 mmol) and stirred at −78° C. for 10 min, warmed to 0° C. for 10 minthen recooled to −78° C. To a separate reaction vessel containingPreparation 1A (3.41 g, 16.07 mmol), also azeotroped twice with benzene,was added toluene (15.3 mL), cooled to −78° C. and LDA (37.0 mL, 18.5mmol) was added, the resulting solution was stirred at −78° for 25 min.At this time the enolate derived from the ester was transferred viacannula into the solution of the oxazolidinone enolate, stirred at −78°C. for an additional 5 min at which time the septum was removed andsolid powdered bis(2-ethylhexanoyloxy)copper (9.02 g, 25.8 mmol) wasrapidly added to the reaction vessel and the septum replaced. The vesselwas immediately removed from the cold bath and immersed into a warmwater bath (40° C.) with rapid swirling with a concomitant color changefrom the initial turquoise to brown. The reaction mixture was stirredfor 20 min, was poured into 5% aqueous NH₄OH (360 mL) and extracted withEtOAc (2×). The combined organics were washed with brine, dried(Na₂SO₄), filtered and concentrated under reduced pressure. The residuewas purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to60% solvent A/B=hexanes/EtOAc, REDISEP® SiO₂ 120 g). Concentration ofappropriate fractions provided Preparation 1C (2.87 g, 66%) as paleyellow viscous oil. ¹H NMR showed the product was a 1.6:1 mixture ofdiastereoisomers 1C:1D as determined by the integration of themultiplets at 2.74 & 2.84 ppm: ¹H NMR (400 MHz, CDCl₃) δ ppm 4.43-4.54(2H, m), 4.23-4.35 (5H, m), 4.01 (1H, ddd, J=9.54, 6.27, 3.51 Hz), 2.84(1H, ddd, J=9.41, 7.28, 3.64 Hz), 2.74 (1H, ddd, J=10.29, 6.27, 4.02Hz), 2.37-2.48 (2H, m, J=10.38, 6.98, 6.98, 3.51, 3.51 Hz), 2.20-2.37(3H, m), 1.92-2.20 (8H, m), 1.64-1.91 (5H, m), 1.47 (18H, s), 0.88-0.98(12H, m).

Preparation 1E:(2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid, and Preparation 1F:(2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid

To a cool (0° C.), stirred solution of Preparation 1C and 1D (4.54 g,9.51 mmol) in THF (140 mL) and water (42 mL) was sequentially addedhydrogen peroxide (30% in water) (10.3 g, 91 mmol) and LiOH (685.3 mg,28.6 mmol) and the mixture was stirred for 1 hr. At this time thereaction vessel was removed from the cold bath and then stirred for 1.5hr. The reaction was judged complete by HPLC. To the reaction mixturewas added saturated NaHCO₃ (45 mL) and saturated Na₂SO₃ (15 mL), andthen partially concentrated under reduced pressure. The resulting crudesolution was extracted with DCM (3×). The aqueous phase was acidified topH˜1-2 with 1N HCl, extracted with DCM (3×) and EtOAc (1×). The combinedorganics were washed with brine, dried (Na₂SO₄), filtered andconcentrated under reduced pressure to provide a mixture of Preparation1E and 1F (3.00 g, 86%) as colorless oil: ¹H NMR (400 MHz, CDCl₃) δ ppm2.76-2.84 (1H, m, diastereoisomer 2), 2.64-2.76 (3H, m), 2.04-2.35 (8H,m), 1.88-2.00 (4H, m), 1.71-1.83 (4H, m), 1.48 (9H, s, diastereoisomer1), 1.46 (9H, s, diastereoisomer 2); ¹H NMR showed a 1.7:1 mixture of1E:1F by integration of the peaks for the t-butyl groups.

Preparation 1E:(2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid, and Preparation 1F:(2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid

To a cold (−78° C.), stirred solution of diisopropylamine (1.7 mL, 11.93mmol) in THF (19 mL) under nitrogen atmosphere was added n-BuLi (2.5M inhexanes) (4.8 mL, 12.00 mmol). The mixture was stirred for 5 min andthen warmed to 0° C. In a separate vessel, to a cold (−78° C.) stirredsolution of the mixture of Preparation 1E and 1F (1.99 g, 5.43 mmol) inTHF (18 mL) was added the LDA solution prepared above via cannula slowlyover 25 min. The mixture was stirred for 15 min, then warmed to roomtemperature (placed in a 24° C. water bath) for 15 min, and then againcooled to −78° C. for 15 min. To the reaction mixture was added Et₂AlCl(1M in hexane) (11.4 mL, 11.40 mmol) via syringe, stirred for 10 min,warmed to room temperature for 15 min and then cooled back to −78° C.for 15 min. Methanol (25 mL) was rapidly added, swirled vigorously whilewarming to room temperature, then concentrated to ˜1/4 original volume.The mixture was dissolved in EtOAc and washed with 1N HCl (50 mL) andice (75 g). The aqueous phase was separated, extracted with EtOAc (2×).The combined organics were washed with a mixture of KF (2.85 g in 75 mLwater) and 1N HCl (13 mL) [resulting solution pH 3-4], then with brine,dried (Na₂SO₄), filtered and concentrated under reduced pressure to givea 9:1 (1E:1F) enriched diastereoisomeric mixture (as determined by ¹HNMR) of Preparation 1E and Preparation 1F (2.13 g, >99%) as a paleyellow viscous oil: ¹H NMR (400 MHz, CDCl₃) δ ppm 2.64-2.76 (2H, m),2.04-2.35 (4H, m), 1.88-2.00 (2H, m), 1.71-1.83 (2H, m), 1.48 (9H, s).

Preparation 1G:(3S)-3-Amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one,and Preparation 1H:(3R)-3-Amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one

Racemic 3-amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one(Rittle, K. E. et al., Tetrahedron Letters, 28(5):521-522 (1987)) wasprepared according to the literature procedure. The enantiomers wereseparated under chiral-SFC conditions using the following method:CHIRALPAK® AS-H 5×25; Mobile phase: 30% MeOH+0.1% DEA in CO₂; Flow rate:280 mL/min; Pressure: 100 bar; Temperature: 35° C.

Obtained the S-enantiomer (Preparation 1G): HPLC: RT=1.75 min (30%MeOH+0.1% DEA in CO₂ on CHIRALPAK® AS-H 4.6×250 mm, 3 mL/min, 35° C.,100 bar, 230 nm, 10 μl injection); ¹H NMR (400 MHz, CDCl₃) δ ppm7.58-7.63 (2H, m), 7.55 (1H, ddd, J=8.50, 7.11, 1.76 Hz), 7.40-7.47 (1H,m), 7.34-7.40 (3H, m), 7.31 (1H, dd, J=7.81, 1.51 Hz), 7.14-7.22 (1H,m), 4.46 (1H, s), 3.44 (3H, s), 3.42 (2H, s); [α]_(D)=−155° (c=1.9,MeOH) (Lit. Rittle, K. E. et al., Tetrahedron Letters, 28(5):521-522(1987): [α]_(D)=−236°).

Also obtained the R-enantiomer (Preparation 1H): HPLC: RT=1.71 min;[α]_(D)=+165° (c=2.1, MeOH) (Lit [α]_(D)=+227°).

Alternate Procedure to Make Preparation 1G

Preparation 1G.CSA salt:(3S)-3-Amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one,(1S)-(+)-10-camphorsulfonic acid salt

Preparation 1G.CSA was prepared from racemic3-amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (9.98g, 37.6 mmol) (prepared according to the literature as shown above)according to the literature procedure (Reider, P. J. et al., J. Org.Chem., 52:955-957 (1987)). Preparation 1G.CSA (16.91 g, 99%) wasobtained as a colorless solid: Optical Rotation: [α]_(D)=−26.99° (c=1,H₂O) (Lit. [α]_(D)=−27.8° (c=1, H₂O))

Preparation 1I: tert-Butyl(2S,3R)-6,6,6-trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate,and Preparation 1J: tert-Butyl(2R,3R)-6,6,6-trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate

To a stirred solution of Preparation 1G (1.45 g, 5.47 mmol) and a 9:1mixture of Preparation 1E and 1F (1.989 g, 5.43 mmol) in DMF (19 mL) wasadded O-benzotriazol-1-yl-N,N,N′,N′-tetra-methyluroniumtetrafluoroborate (1.79 g, 5.57 mmol) and triethylamine (3.0 mL, 21.52mmol) and stirred overnight. The reaction was judged complete by LCMS.The reaction mixture was poured into water (125 mL) and the precipitatedsolid was collected by filtration, washed with water and air dried toprovide an 8:1 mixture of Preparation 1I and Preparation 1J (2.95 g,89%) as a cream solid: MS (ES): m/z=614 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃)δ ppm 7.55-7.65 (3H, m), 7.44-7.52 (2H, m), 7.35-7.45 (4H, m), 5.52 (1H,d, J=8.03 Hz), 3.48 (3H, s), 2.63 (2H, ddd, J=9.35, 3.95, 3.76 Hz),2.14-2.25 (4H, m), 1.90-2.03 (3H, m), 1.69-1.82 (1H, m), 1.51 (9H, s).

Preparation 1K:(2S,3R)-6,6,6-Trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoicacid, and Preparation 1L:(2R,3R)-6,6,6-Trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoicacid

To a cool (0° C.), stirred solution of the above mixture of Preparation1I and Preparation 1J (2.95 g, 4.81 mmol) in DCM (20 mL) was added TFA(20 mL, 260 mmol). The reaction mixture was stirred for 1 hr, thenallowed to warm to room temperature and stirred for 2.5 hr. The reactionwas judged complete by LCMS. The reaction mixture was diluted withtoluene (50 mL) and concentrated under reduced pressure. The residuemixture was redissolved in toluene (50 mL) and concentrated underreduced pressure then dried under high vacuum. The crude product wasdissolved in DCM, SiO₂ (15 g) was added, concentrated, then was purifiedby flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 45% solventA/B=DCM/EtOAc, REDISEP® SiO₂ 80 g). Concentration of appropriatefractions provided a mixture of Preparation 1K and Preparation 1L (2.00g, 75%) as a cream solid: HPLC: RT=2.770 min (CHROMOLITH® SpeedROD4.6×50 mm (4 min grad) eluting with 10-90% aqueous MeOH over 4 minutescontaining 0.1% TFA, 4 mL/min, monitoring at 254 nm); MS (ES): m/z=558[M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ ppm 8.32 (1H, d, J=8.03 Hz), 7.65-7.71(1H, m), 7.50-7.60 (3H, m), 7.41-7.49 (2H, m), 7.39 (1H, dd, J=7.91,1.63 Hz), 7.23-7.35 (2H, m), 5.59 (1H, d, J=8.03 Hz), 3.51 (3H, s), 2.81(1H, ddd, J=10.54, 6.90, 3.64 Hz), 2.67-2.76 (1H, m), 2.22-2.33 (3H, m),1.99-2.12 (3H, m), 1.85-1.94 (1H, m), 1.79 (1H, ddd, J=13.87, 7.84, 3.64Hz).

Example 1

To a stirred solution of an 8:1 mixture of Preparation 1K andPreparation 1L (3.46 g, 6.21 mmol) in DMF (25 mL) under nitrogenatmosphere was added ammonium chloride (3.32 g, 62.1 mmol), EDC (3.55 g,18.52 mmol), HOBT (2.85 g, 18.61 mmol), and triethyl amine (16 mL, 115mmol) and stirred overnight. The reaction was judged complete by LCMS.The reaction mixture was poured into water (200 mL) with vigorousswirling and then allowed to sit. The solid was collected by filtration,washed with water, allowed to dry to afford 3.6 g colorless solid. Thesolid was purified by preparative SFC chromatography (Lux-Cellulose-2(3×25 cm), 8% methanol in CO₂, 140 ml/min @220 nm and 35° C.; Sample:3.6 g in 50 cc methanol, conc.=70 mg/ml, Stack injection: 0.5 cc/9.2min). Fractions containing product were concentrated, dried overnightunder vacuum. Obtained Example 1 (2.74 g, 79%) as a colorless solid(Crystal Form N-1): HPLC: RT=9.601 min (H₂O/CH₃CN with TFA, Sunfire C183.5 um, 4.6×150 mm, 4.6×150 mm, gradient=15 min, wavelength=220 and 254nm). MS (ES): m/z=557 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.54 (1H,d, J=7.28 Hz), 7.71-7.80 (1H, m), 7.68 (2H, d, J=8.78 Hz), 7.50-7.62(3H, m), 7.45 (2H, t, J=7.28 Hz), 7.29-7.40 (2H, m), 7.15 (1H, br. s.),5.30 (1H, d, J=7.28 Hz), 3.39 (3H, s), 2.74-2.86 (1H, m), 2.02-2.32 (3H,m), 1.45-1.79 (4H, m); [α]_(D)=−107.0° (5.73 mg/mL, DMSO).

Crystal Form A-2 was prepared by adding approximately 1 mg of Example 1to approximately 0.7 mL of acetone/acetonitrile/water solution (2:2:1).A mixture of colorless needles and thin blades crystals were obtainedafter one day of slow evaporation of the solution at room temperature.The thin blade crystals were separated to provide crystal Form A-2.

Crystal Form EA-3 was prepared by adding approximately 1 mg of Example 1to approximately 0.7 mL of ethyl acetate/heptane solution (1:1).Colorless blade crystals were obtained after three days of slowevaporation of the solution at room temperature.

Crystal Form THF-2 was obtained by adding approximately 5 mg of Example1 to approximately 0.7 mL of THF/water solution (4:1). Colorlessblade-like crystals were obtained after one day of solvent evaporationat room temperature.

Alternate Procedure to Make Example 1 Preparation 1M:3,3,3-Trifluoropropyl trifluoromethanesulfonate

To a cold (−25° C.), stirred solution of 2,6-lutidine (18.38 mL, 158mmol) in CH₂Cl₂ (120 mL) was added Tf₂O (24.88 mL, 147 mmol) over 3 min,and stirred for 5 min. To the reaction mixture was added3,3,3-trifluoropropan-1-ol (12 g, 105 mmol) over an interval of 3 min.After 2 hr, the reaction mixture was warmed to room temperature andstirred for 1 hr. The reaction mixture was concentrated to half volume,then purified by loading directly on silica gel column (330 g ISCO) andeluted with CH₂Cl₂. Obtained Preparation 1M (13.74 g, 53%) as acolorless oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 4.71 (2H, t, J=6.15 Hz),2.49-2.86 (2H, m).

Preparation 1N:(4S)-4-Benzyl-3-(5,5,5-trifluoropentanoyl)-1,3-oxazolidin-2-one

Preparation 1N was prepared from 5,5,5-trifluoropentanoic acid (3.35 g,21.46 mmol) and (4S)-4-benzyl-1,3-oxazolidin-2-one (3.80 g, 21.46 mmol)by the general methods shown for Preparation 1B. Preparation 1N (5.67 g,84%) was obtained as a colorless viscous oil: ¹H NMR (400 MHz, CDCl₃) δppm 7.32-7.39 (2H, m), 7.30 (1H, d, J=7.05 Hz), 7.18-7.25 (2H, m),4.64-4.74 (1H, m), 4.17-4.27 (2H, m), 3.31 (1H, dd, J=13.35, 3.27 Hz),3.00-3.11 (2H, m), 2.79 (1H, dd, J=13.35, 9.57 Hz), 2.16-2.28 (2H, m),1.93-2.04 (2H, m).

Preparation 1O: tert-Butyl(3R)-3-(4S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl)carbonyl)-6,6,6-trifluorohexanoate

To a cold (−78° C.), stirred solution of Preparation 1N (3.03 g, 9.61mmol) in THF (20 mL) was added NaHMDS (1.0M in THF) (10.6 mL, 10.60mmol) under nitrogen atmosphere. After 2 hours, tert-butyl2-bromoacetate (5.62 g, 28.8 mmol) was added neat via syringe at −78° C.and stirring was maintained at the same temperature. After 6 hours, thereaction mixture was warmed to room temperature. The reaction mixturewas partitioned between saturated NH₄Cl and EtOAc. The organic phase wasseparated, and the aqueous was extracted with EtOAc (3×). The combinedorganics were washed with brine, dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography (Teledyne ISCO CombiFlash Rf, 5% to 100% solventA/B=hexanes/EtOAc, REDISEP® SiO₂ 120 g). Concentration of appropriatefractions provided Preparation 1O (2.79 g, 67.6%) as a colorless viscousoil: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.34 (2H, d, J=7.30 Hz), 7.24-7.32(3H, m), 4.62-4.75 (1H, m, J=10.17, 6.89, 3.43, 3.43 Hz), 4.15-4.25 (3H,m), 3.35 (1H, dd, J=13.60, 3.27 Hz), 2.84 (1H, dd, J=16.62, 9.57 Hz),2.75 (1H, dd, J=13.35, 10.07 Hz), 2.47 (1H, dd, J=16.62, 4.78 Hz),2.11-2.23 (2H, m), 1.90-2.02 (1H, m), 1.72-1.84 (1H, m), 1.44 (9H, s).

Preparation 1P:(2R)-2-(2-tert-Butoxy-2-oxoethyl)-5,5,5-trifluoropentanoic acid

Preparation 1P was prepared from Preparation 1O (2.79 g, 6.50 mmol) bythe general methods shown for Preparation 1E. Preparation 1P (1.45 g,83%) was obtained as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ ppm2.83-2.95 (1H, m), 2.62-2.74 (1H, m), 2.45 (1H, dd, J=16.62, 5.79 Hz),2.15-2.27 (2H, m), 1.88-2.00 (1H, m), 1.75-1.88 (1H, m), 1.45 (9H, s).

Preparation 1E:(2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid, and Preparation 1F:(2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid

To a cold (−78° C.), stirred solution of Preparation 1P (5.44 g, 20.13mmol) in THF (60 mL) was slowly added LDA (24.60 mL, 44.3 mmol) over 7min. After stirring for 2 hr, Preparation 1M (6.44 g, 26.2 mmol) wasadded to the reaction mixture over 3 min. After 45 min, the reactionmixture was warmed to −25° C. bath (ice/MeOH/dry ice) for 1 hr, and thenwarmed to 0° C. After 45 min, Preparation 1M (1 g) was added and thereaction mixture was stirred for 20 min. The reaction was quenched withwater and 1N NaOH and was extracted with CH₂Cl₂. The organic layer wasagain extracted with 1N NaOH (2×) and the aqueous layers were combined.The aqueous layer was cooled in ice/water bath and then acidified withconcentrated HCl to pH 2. Next, the aqueous layer was extracted withEtOAc. The combined organics were washed with brine, dried overanhydrous sodium sulphate, and concentrated under reduced pressure. Theresidue was dried under high vacuum to provide a 1:5 (1E:1F) mixture (asdetermined by ¹H NMR) of Preparation 1E and Preparation 1F (5.925 g,80%) as a pale yellow solid. ¹H NMR (500 MHz, CDCl₃) δ ppm 2.81 (1H,ddd, J=10.17, 6.32, 3.85 Hz), 2.63-2.76 (1H, m), 2.02-2.33 (4H, m),1.86-1.99 (2H, m), 1.68-1.85 (2H, m), 1.47 (9H, s).

Preparation 1E:(2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid, and Preparation 1F:(2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid

A mixture of Preparation 1E and Preparation 1F (64 mg, 1.758 mmol) wastaken in THF (6 mL) to give a colorless solution which was cooled to−78° C. Then, LDA (2.149 mL, 3.87 mmol) (1.8M inheptane/THF/ethylbenzene) was slowly added to the reaction mixture over10 min. After stirring for 15 min the reaction mixture was placed in aroom temperature water bath. After 15 min the reaction mixture wasplaced back in −78° C. bath and then diethylaluminum chloride (3.87 mL,3.87 mmol) (1M in hexane) was added slowly over 5 min. The reactionmixture was stirred at −78° C. After 15 min the reaction mixture wasplaced in a room temperature water bath for 10 min and then cooled backto −78° C. bath. After 15 min the reaction was quenched with MeOH (8 mL,198 mmol), removed from the −78° C. bath and concentrated. To thereaction mixture was added ice and HCl (16 mL, 16.00 mmol), followed byextraction with EtOAc (2×). The organic layer was washed with potassiumfluoride (920 mg, 15.84 mmol) (in 25 mL H₂O) and HCl (4.5 mL, 4.50mmol). The organics were dried over anhydrous magnesium sulphate andconcentrated under reduced pressure to provide a 9:1 (1E:1F) enrichedmixture of Preparation 1E and Preparation 1F (540 mg, 1.583 mmol, 90%yield) as light yellow/orange solid. ¹H NMR (400 MHz, CDCl₃) δ ppm2.64-2.76 (2H, m), 2.04-2.35 (4H, m), 1.88-2.00 (2H, m), 1.71-1.83 (2H,m), 1.48 (9H, s). It was converted to Example 1 by the sequence ofreactions as outlined above.

Alternate Procedure to Make Preparation 1E Preparation 1Q:(2R,3S)-1-Benzyl 4-tert-butyl 2,3-bis(3,3,3-trifluoropropyl)succinate

A clean and dry 5 L four neck round bottom flask equipped withmechanical stirring, thermometer socket and nitrogen bubbler at roomtemperature was charged with N,N-dimethyl formamide (2.07 L), a 1.2:1mixture of Preparation 1E and Preparation 1F (207 g, 0.5651 moles),potassium carbonate (117.1 g, 0.8476 moles) followed by benzyl bromide(116 g, 0.6781 moles) over 15-20 min. The reaction mixture was stirredfor 2-3 hr. After completion of the reaction, the reaction mixture wasconcentrated to dryness at 50-55° C. under vacuum. Ethyl acetate (3.1 L,30 Vol.) was charged into the concentrated reaction mass and then washedwith water (2.07 L), brine (0.6 L) then dried over anhydrous sodiumsulfate (207 g), filtered and concentrated to dryness at 40-45° C. undervacuum. The residue was dissolved in dichloromethane (1.035 L, 5 vol.)and then absorbed onto silica gel (60-120) (607 g, 3.0 w/w), then waspurified with column chromatography using petroleum ether and ethylacetate as solvents. After pooling several batches, Preparation 1Q (235g) was obtained. HPLC purity: 99.77%,

Preparation 1E:(2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid

A clean and dry 2 L autoclave was charged with methanol (540 mL) and waspurged with nitrogen for 5-10 minutes. To the autoclave was added 10%palladium on carbon (12 g, 20%), purged with nitrogen once again for5-10 min then was charged with Preparation 1Q (60 g, 0.1315 moles), theautoclave was flushed with methanol (60 mL) and stirred for 4-6 hr at20-25° C. under 5 Kg hydrogen pressure. After completion of thereaction, the reaction mass was filtered through CELITE®, washed withmethanol (180 mL), dried with anhydrous sodium sulfate (60 g), filteredand concentrated to dryness at 45-50° C. under vacuum. ObtainedPreparation 1E (45.8 g, 95%) as a colorless solid: HPLC purity: 98.9%.

Alternate Procedure to Make Preparation 1E Preparation 1E:(2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoicacid

Preparation 1E was prepared in a procedure identical as above from amixture of Preparations 1E and 1F (200 g, 0.5460 moles) using LDA (1.8 Msolution in THF, ethyl benzene and heptane) (698 mL, 2.3 equiv.) anddiethyl aluminum chloride (1.0 M solution in hexane) (1256 mL, 2.3equiv) in THF (2.0 L). After workup as explained above, the resultingresidue was treated as follows: The crude material was added to a 2Lfour neck round bottom flask, followed by the addition of MTBE (1.0 L)charged below 30° C. The resulting mixture was stirred for 5-10 minutesto obtain a clear solution. Hexanes (600 mL) was charged to the reactionmixture at a temperature below 30° C. The reaction mixture was stirredfor 10 min. Next, tert-butylamine (43.8 g, 1.1 eq) was charged slowlyover a period of 15 minutes below 30° C. This addition was observed tobe exothermic. The reaction mixture was stirred for 2 hrs below 30° C.and filtered. The solid material was washed with 5:3 MTBE:hexane (200mL), the filtrate was concentrated and transferred to an amber colorbottle. The filtered solid was dissolved in dichloromethane (2.0 L),washed with 1N HCl (2.0), the organic layer was washed with brine(1.0L×2), then was concentrated under reduced pressure below 45° C. Thismaterial was found to be 91.12% pure. The material was repurified by theabove t-butylamine crystallization purification procedure. ObtainedPreparation 1E (78 g, 39%): HPLC purity: 99.54%.

Alternate Procedure to Make Example 1 Preparation 1I: tert-Butyl(2S,3R)-6,6,6-trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate

A clean and dry 2 L four neck round bottom flask equipped withmechanical stirring, thermometer socket and nitrogen bubbler was chargedwith N,N-dimethylformamide (457 mL), Preparation 1E (45.7 g, 0.1248moles) and Preparation 1G.CSA (62.08 g, 0.1248 moles) under nitrogenatmosphere at 20-25° C. The reaction mixture was stirred for 15-20minutes to make clear solution at 20-25° C. To the reaction mixture wasadded TBTU (48.16 g, 0.1498 moles) at 20-25° C. followed bytriethylamine (50.51 g, 0.4992 moles) over 15-20 minutes at 20-25° C.The reaction mixture was stirred for 60-120 minutes at 20-25° C. undernitrogen atmosphere. After completion of the reaction, the reaction wasquenched into water (1.37L, 30 Vol.) at 20-25° C. under stirring. Thereaction mixture was stirred for 30 minutes at 20-25° C. The reactionmixture was filtered and washed with water (228 mL). The resulting solidmaterial was dissolved in ethyl acetate (457 mL), washed with water(2×137 mL), brine (137 mL), and then dried with anhydrous sodium sulfate(45.7 g). Activated charcoal (9.14 g, 20%) was charged into the reactionmixture and stirred for 30 minutes. The mixture was filtered throughCELITE® bed and 1 micron filter cloth, washed charcoal bed with ethylacetate (137 mL), concentrated to 1.0 Vol. stage and then petroleumether (457 mL, 10 Vol.) was charged and stirred for 30 minutes at 20-25°C. The solid was collected by filtration, washed with petroleum ether(137 mL) and then dried under vacuum at 40-45° C. for 8 hr until loss ondrying was less than 3.0%. Obtained Preparation 1I (65.2 g, 85%): HPLCpurity: 98.26%.

Preparation 1K:(2S,3R)-6,6,6-Trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoicacid

A clean and dry 3 L four neck round bottom flask equipped withmechanical stirring, thermometer socket and nitrogen bubbler was chargedwith dichloromethane (980 mL) under nitrogen atmosphere followed byPreparation 1I (140 g, 0.2282 moles) at 20-25° C. The reaction mixturewas cooled to 0-5° C. and trifluoroacetic acid (980 mL) was chargedslowly for 30-40 minutes. The resulting mixture was stirred for 2 hr at0-5° C. under nitrogen atmosphere. The reaction temperature was raisedto 20 to 25° C., and the reaction mixture was stirred for 1-2 hr at 20to 25° C. After completion of the reaction, the reaction mixture wasconcentrated to dryness at 50 to 55° C. under vacuum. Toluene (3×700mL,) was charged into the concentrated reaction mass, and then distilledoff at 50 to 55° C. under vacuum. After complete concentration fromtoluene, ethyl acetate (280 mL) was charged into the reaction mass at 20to 25° C., stirred for 60 minutes, then the solid was collected byfiltration, washed with ethyl acetate (140 mL), dried under vacuum at 50to 55° C. for 12 hr until loss on drying was less than 2.0%. ObtainedPreparation 1K (106 g, 84%): HPLC purity: 98.43%.

Example 1

A reaction vessel was charged with Preparation 1K (30 g, 53.81 mmol),HOBt (8.7 g, 64.38 mmol), and THF (150 mL) at room temperature. To thehomogeneous solution was added EDCI (12.4 g, 64.68 mmol), stirred for 15min, then cooled to 8° C. To the reaction mixture was added ammonia (2Min IPA) (81 mL, 162 mmol) over 5 min so as to maintain a temperaturebelow 10° C. The resulting heavy slurry was stirred for 10 min, warmedto room temperature over 30 min, then stirred for 4 hr. At thecompletion of the reaction, water (230 mL) was slowly added over 15 minto maintain a temperature below 20° C., and then stirred for 2 hr. Thesolid was collected by filtration, washed with water (3×60 mL), thendried under vacuum 48 hr at 55° C. The above crude product was chargedinto a 1 L 3-necked round flask. IPA (200 mL) was added, then heated to80° C. resulting in a homogeneous solution. Water (170 mL) was slowlyadded (15 min) to maintain an internal temperature >75° C. The resultingslurry was stirred and cooled to room temperature for 2 hr. The solidwas collected by filtration, washed with water (2×50 mL), then driedunder vacuum (55° C. for 24 h, and 30° C. for 48 h). Obtained Example 1(23.4 g, 78% yield): HPLC purity: 99.43%.

Example 2(2R,3S)—N-((3S)-2-Oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide

Preparation 2A:(3S)-3-Amino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, andPreparation 2B:(3R)-3-Amino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one

Racemic 3-amino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (J. Med.Chem., 49:2311-2319 (2006), compound#5) was prepared according to theliterature procedure. The enantiomers were separated on Berger SFC MGIIIColumn: Lux 25×3 cm, 5 cm; Mobile phase: 30% MeOH+0.1% DEA in CO₂; Flowrate: 150 mL/min; Temperature: 40° C.; Detector wavelength: 250 nM.Obtained the S-enantiomer Preparation 2A as a white solid: ¹H NMR (400MHz, DMSO-d₆) δ ppm 10.67 (1H, br. s.), 7.58 (1H, td, J=7.65, 1.76 Hz),7.37-7.53 (5H, m), 7.23-7.30 (2H, m), 7.14-7.22 (1H, m), 4.23 (1H, s),2.60 (2H, br. s.); HPLC: RT=3.0625 min (30% MeOH+0.1% DEA in CO₂ on OD-HColumn, 3 mL/min, 35° C., 96 bar, 230 nm, 10 μl inj); [α]_(D)=−208.3°(5.05 mg/mL, MeOH). Also obtained the R-enantiomer Preparation 2B as anoff white solid: HPLC: RT=3.970 min; [α]_(D)=182.1° (2.01 mg/mL, MeOH).

Preparation 2C: tert-Butyl(2S,3R)-6,6,6-trifluoro-3-(((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate,and Preparation 2D: tert-Butyl(2R,3R)-6,6,6-trifluoro-3-(((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate

Preparation 2C was prepared from Preparation 2A (564 mg, 2.244 mmol) anda mixture of Preparation 1E and Preparation 1F (822 mg, 2.244 mmol)according to the general procedure shown for Preparation 1I. ObtainedPreparation 2C and Preparation 2D (1.31 g, 97%): HPLC: RT=3.443 min(CHROMOLITH® ODS 4.6×50 mm (4 min grad) eluting with 10-90% aqueous MeOHover 4 minutes containing 0. % TFA, 4 mL/min, monitoring at 220 nm); MS(ES): m/z=600.3 [M+H]⁺.

Preparation 2E:(2S,3R)-6,6,6-Trifluoro-3-(((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoicacid, and Preparation 2F:(2R,3R)-6,6,6-Trifluoro-3-(((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoicacid

A mixture of Preparation 2E and Preparation 2F was prepared from amixture of Preparation 2C and Preparation 2D (1.31 g, 2.185 mmol) by thegeneral methods shown for Preparation 1K. Obtained a mixture ofPreparation 2E and Preparation 2F (1.18 g, 99%): HPLC: RT=2.885 min(CHROMOLITH® ODS 4.6×50 mm (4 min grad) eluting with 10-90% aqueous MeOHover 4 minutes containing 0. % TFA, 4 mL/min, monitoring at 220 nm). MS(ES): m/z=544.2 [M+H]⁺.

Example 2

Example 2 was prepared from a mixture of Preparation 2E and Preparation2F (354 mg, 0.651 mmol) by the general methods shown for Example 1.After separation of the diastereoisomers, Example 2 was obtained (188mg, 52%) as a white solid: HPLC: RT=9.063 min (H₂O/CH₃CN with TFA,Sunfire C18 3.5 um, 4.6×150 mm, 4.6×150 mm, gradient=15 min,wavelength=220 and 254 nm); MS (ES): m/z=543 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d₆) δ ppm 10.87 (1H, br. s.), 9.50-9.55 (1H, m), 7.62-7.69 (2H, m),7.40-7.57 (5H, m), 7.29-7.36 (2H, m), 7.22-7.28 (1H, m), 7.16 (1H, br.s.), 5.25 (1H, d), 3.30-3.32 (1H, m), 2.75-2.86 (1H, m), 2.44-2.48 (1H,m), 2.06-2.34 (3H, m), 1.51-1.77 (4H, m); [α]_(D)=−114.4° (8.04 mg/mL,DMSO).

Crystal Form M2-1 was prepared by adding approximately 1 mg of Example 2to approximately 0.7 mL of MeOH/fluorobenzene solution (3:1). Colorlessplate-like crystals were obtained after 2 days of solvent evaporation atroom temperature.

Example 3(2R,3S)—N-((3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2-(2,2,2-trifluoroethyl)-3-(3,3,3-trifluoropropyl)succinamide

Preparation 3A:(4S)-4-(Propan-2-yl)-3-(4,4,4-trifluorobutanoyl)-1,3-oxazolidin-2-one

Preparation 3A was prepared from(4S)-4-(propan-2-yl)-1,3-oxazolidin-2-one (4.66 g, 36.1 mmol) and4,4,4-trifluorobutanoic acid (5.02 g, 35.3 mmol) by the general methodsshown for Preparation 1B. Preparation 3A was obtained as a colorless oil(3.64 g, 40%). ¹H NMR (400 MHz, CDCl₃) δ ppm 4.44 (1H, ddd, J=8.41,3.51, 3.39 Hz), 4.31 (1H, t, J=8.66 Hz), 4.25 (1H, dd, J=9.03, 3.26 Hz),3.13-3.32 (2H, m), 2.47-2.59 (2H, m), 2.38 (1H, dddd, J=13.96, 7.01,3.89 Hz), 0.93 (3H, d, J=7.28 Hz), 0.88 (3H, d, J=6.78 Hz).

Preparation 3B: tert-Butyl(3R)-5,5,5-trifluoro-3-(((4S)-2-oxo-4-(propan-2-yl)-1,3-oxazolidin-3-yl)carbonyl)-2-(3,3,3-trifluoropropyl)pentanoate

Preparation 3B was prepared from Preparation 3A (1.04 g, 4.12 mmol) andtert-butyl 5,5,5-trifluoropentanoate (Preparation 1A) (1.55 g, 7.28mmol) by the general methods shown for Preparation 1C. Preparation 3B(528.3 mg, 28%) was obtained as a pale yellow viscous oil: ¹H NMR (400MHz, CDCl₃) δ ppm 4.57 (1H, ddd, J=10.54, 5.02, 1.76 Hz), 4.41-4.50 (2H,m), 4.20-4.32 (4H, m), 2.77-2.88 (3H, m), 2.70 (1H, dt, J=9.79, 4.89Hz), 2.38 (1H, dddd, J=10.38, 6.87, 3.64, 3.45 Hz), 2.23-2.34 (4H, m),2.06-2.18 (2H, m), 1.93-2.05 (2H, m), 1.69-1.81 (4H, m), 1.46 (9H, s),1.43 (9H, s), 0.85-0.97 (12H, m).

Preparation 3C:(2R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(2,2,2-trifluoroethyl)hexanoicacid

Preparation 3C was prepared from Preparation 3B (528.3 mg, 1.140 mmol)by the general methods shown for Preparation 1D. Preparation 3C (306.7mg, 76%) was obtained as a colorless waxy solid (306.7 mg, 76%). ¹H NMR(400 MHz, CDCl₃) δ ppm 3.08 (1H, ddd, J=8.72, 4.20, 3.89 Hz), 3.00 (1H,ddd, J=9.66, 7.03, 2.89 Hz), 2.70-2.82 (4H, m), 2.36 (1H, ddd, J=15.25,10.73, 3.64 Hz), 2.18-2.30 (2H, m), 2.12 (2H, dd, J=10.16, 5.65 Hz),1.90-2.02 (2H, m), 1.70-1.81 (3H, m), 1.45-1.51 (18H, m).

Preparation 3D:(2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(2,2,2-trifluoroethyl)hexanoicacid

Preparation 3D was prepared from Preparation 3C (306.7 mg, 0.871 mmol)by the general methods shown to enrich the mixture of Preparation 1E andPreparation 1F. Preparation 3D (297.0 mg, 97%) was obtained as a yellowwaxy solid (297.0 mg, 97%). ¹H NMR (400 MHz, CDCl₃) δ ppm 2.99 (1H, ddd,J=9.47, 6.96, 2.89 Hz), 2.69-2.82 (2H, m), 2.18-2.31 (2H, m), 2.06-2.18(1H, m), 1.91-2.03 (1H, m), 1.68-1.80 (1H, m), 1.46-1.51 (9H, m).

Preparation 3E: tert-Butyl(2S,3R)-5,5,5-trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)pentanoate

Preparation 3E was prepared from Preparation 3D (297.0 mg, 0.843 mmol)and Preparation 1G (212.0 mg, 0.799 mmol) by the general methods shownfor Preparation 1I. Preparation 3E (471.7 mg, 98%) was obtained as a tansolid (471.7 mg, 98%). MS (ES): m/z=600 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃)δ ppm 7.75 (1H, d, J=7.78 Hz), 7.54-7.64 (3H, m), 7.43-7.51 (1H, m),7.34-7.43 (4H, m), 7.22-7.31 (1H, m), 5.50 (1H, d, J=7.53 Hz), 3.48 (3H,s), 2.87-2.96 (1H, m), 2.73-2.83 (1H, m), 2.60 (1H, td, J=10.10, 3.89Hz), 2.13-2.25 (3H, m), 1.86-2.05 (2H, m), 1.52 (9H, s).

Preparation 3F:(2S,3R)-5,5,5-Trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)pentanoicacid

Preparation 3F was prepared from Preparation 3E (466.1 mg, 0.777 mmol)by the general methods shown for Preparation 1H. Preparation 3F (451mg, >99%) was obtained as a tan solid. MS (ES): m/z=544 [M+H]⁺; ¹H NMR(400 MHz, CDCl₃) δ ppm 8.29 (1H, d, J=7.53 Hz), 7.64 (1H, td, J=7.84,1.63 Hz), 7.53-7.60 (2H, m), 7.49 (1H, t, J=7.40 Hz), 7.33-7.46 (4H, m),7.22-7.33 (2H, m), 5.53 (1H, d, J=7.53 Hz), 3.49 (3H, s), 3.04-3.21 (2H,m), 2.69-2.81 (2H, m), 2.23-2.33 (2H, m), 2.07-2.19 (2H, m).

Example 3

Example 3 was prepared from Preparation 3F (446 mg, 0.821 mmol) by thegeneral methods shown for Example 1. After separation of thediastereoisomers, Example 3 was obtained: HPLC: RT=3.17 min (H₂O/CH₃CNwith TFA, Sunfire C18 3.5 um, 4.6×150 mm, 4.6×150 mm, gradient=15 min,wavelength=220 and 254 nm). MS (ES): m/z=543.3 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d₆) δ ppm 9.72 (1H, d, J=8.53 Hz), 7.71-7.77 (1H, m), 7.66-7.71(1H, m), 7.64 (1H, d, J=1.25 Hz), 7.48-7.57 (3H, m), 7.39-7.47 (2H, m),7.30-7.39 (2H, m), 7.23 (1H, s), 5.36 (1H, d, J=8.53 Hz), 3.39 (3H, s),3.12-3.23 (1H, m), 2.53-2.61 (1H, m), 2.43 (1H, td, J=10.10, 3.89 Hz),2.16-2.28 (1H, m), 2.02-2.16 (1H, m), 1.82-1.96 (1H, m), 1.68-1.82 (1H,m).

Example 4(2R,3S)—N-((3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-3-(2,2,2-trifluoroethyl)-2-(3,3,3-trifluoropropyl)succinamide

Preparation 4A: tert-Butyl 4,4,4-trifluorobutanoate

Preparation 4A was prepared from 4,4,4-trifluorobutanoic acid (4.99 g,35.1 mmol) using the general procedure shown for Preparation 1A.Preparation 4A (5.58 g, 80%) was obtained as a colorless oil. ¹H NMR(400 MHz, CDCl₃) δ ppm 2.47-2.52 (2H, m), 2.37-2.45 (2H, m), 1.46 (9H,s).

Preparation 4B: tert-Butyl(3R)-6,6,6-trifluoro-3-(((4S)-2-oxo-4-(propan-2-yl)-1,3-oxazolidin-3-yl)carbonyl)-2-(2,2,2-trifluoroethyl)hexanoate

Preparation 4B was prepared from Preparation 4A (1058.3 mg, 5.34 mmol)and Preparation 1B (809.2 mg, 3.03 mmol) according to the generalprocedure shown for Preparation 1C. Preparation 4B (690.1 mg, 49%) wasobtained as a pale yellow viscous oil. ¹H NMR (400 MHz, CDCl₃) δ ppm4.45-4.52 (1H, m), 4.23-4.40 (1H, m), 4.05-4.12 (1H, m), 3.70 (1H, t,J=6.7 Hz), 3.05 (1H, ddd, J=9.9, 5.0, 2.3 Hz), 2.99 (1H, ddd, J=11.2,5.8, 1.8 Hz), 2.58-2.91 (2H, m), 2.38-2.49 (1H, m), 2.27-2.36 (1H, m),2.07-2.26 (1H, m), 1.96-2.04 (1H, m), 1.85-1.94 (1H, m), 1.72-1.82 (1H,m), 1.46 (3H, s), 0.88-0.98 (3H, m); HPLC: RT=3.36 min (MeOH/H₂O/0.1%TFA, CHROMOLITH® SpeedROD 4.6×50 mm, 4 min gradient, wavelength=220 nm).

Preparation 4C:(2R)-3-(tert-Butoxycarbonyl)-5,5,5-trifluoro-2-(3,3,3-trifluoropropyl)pentanoicacid

Preparation 4C was prepared from Preparation 4B (690.1 mg, 1.489 mmol)according to the general procedure shown for Preparation 1E. Preparation4C (335.9 mg, 64%) was obtained as a colorless oil. ¹H NMR (400 MHz,CDCl₃) δ ppm 2.91-3.03 (1H, m), 2.63-2.88 (2H, m), 2.50 (1H, t, J=7.3Hz), 2.07-2.43 (4H, m), 1.89-2.05 (2H, m), 1.73-1.88 (1H, m), 1.47 (5H,s), 1.47 (4H, s).

Preparation 4D:(2R,3S)-3-(tert-Butoxycarbonyl)-5,5,5-trifluoro-2-(3,3,3-trifluoropropyl)pentanoicacid

Preparation 4D was prepared from Preparation 4C (335.9 mg, 0.954 mmol)according to the general procedure shown to enrich the mixture ofPreparation 1E and Preparation 1F. Preparation 4D (277.8 mg, 83%) wasobtained as a brown oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.90-3.03 (1H,m), 2.64-2.88 (2H, m), 2.50 (1H, t, J=7.3 Hz), 2.06-2.43 (3H, m),1.89-2.03 (1H, m), 1.73-1.88 (1H, m), 1.47 (9H, s).

Preparation 4E: tert-Butyl(2S,3R)-6,6,6-trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(2,2,2-trifluoroethyl)hexanoate

Preparation 4E was prepared from Preparation 4D (277.8 mg, 0.789 mmol)and Preparation 1G (210.2 mg, 0.792 mmol) according to the generalprocedure shown for Preparation 1I. Preparation 4E was obtained as acream solid (420.2 mg, 89%). ¹H NMR (400 MHz, CDCl₃) δ ppm 7.55-7.65(3H, m), 7.48 (1H, d, J=7.5 Hz), 7.36-7.45 (4H, m), 5.51 (1H, d, J=7.8Hz), 3.49 (3H, s), 2.87-2.92 (1H, m), 2.63 (1H, s), 2.47-2.58 (1H, m),2.16-2.36 (2H, m), 1.93-2.06 (1H, m), 1.80 (1H, s), 1.51 (9H, s); LCMS:RT=4.02 min (MeOH/H₂O/0.1% TFA, CHROMOLITH® RP-18e 2.0×50 mm, 4 mingradient, wavelength=254 nm); MS (ES):m/z=600 [M+H⁺].

Preparation 4F:(2S,3R)-6,6,6-Trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(2,2,2-trifluoroethyl)hexanoicacid

Preparation 4F was prepared from Preparation 4E (417.2 mg, 0.696 mmol)according to the general procedure shown for Preparation 1K. Preparation4F was obtained as a TFA solvate as an amber solid (476.0 mg, 96%). ¹HNMR (400 MHz, CDCl₃) δ ppm 8.25 (1H, d, J=8.0 Hz), 7.73-7.82 (1H, m),7.56-7.67 (2H, m), 7.34-7.54 (3H, m), 5.67 (1H, d, J=8.3 Hz), 3.49-3.60(2H, m), 3.05-3.13 (1H, m), 2.81-2.97 (1H, m), 2.39-2.60 (1H, m),2.18-2.33 (1H, m), 1.95-2.13 (1H, m); LCMS: RT=3.43 min (MeOH/H₂O/0.1%TFA, CHROMOLITH® RP-18e 2.0×50 mm, 4 min gradient, wavelength=254 nm);MS (ES):m/z=544 [M+H⁺].

Example 4

Example 4 was prepared from Preparation 4F (476.3 mg, 0.667 mmol)according to the general procedure shown for Example 1. After separationof the diastereoisomers, Example 4 was obtained as a cream solid (120.3mg, 32%). HPLC: RT=9.446 min (H₂O/CH₃CN with TFA, Sunfire C18 3.5 μm,4.6×150 mm, 4.6×150 mm, gradient=15 min, wavelength=220 and 254 nm); MS(ES):m/z=543 [M+H⁺]; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.56 (1H, d, J=7.03Hz), 7.82 (1H, s), 7.70-7.78 (1H, m), 7.64-7.70 (1H, m), 7.50-7.63 (3H,m), 7.47 (2H, d, J=7.78 Hz), 7.30-7.42 (2H, m), 7.21 (1H, s), 5.30 (1H,d, J=7.03 Hz), 3.39 (3H, s), 2.67-2.80 (2H, m), 2.51-2.62 (2H, m),2.19-2.29 (1H, m), 2.07-2.21 (1H, m), 1.60-1.72 (2H, m).

Example 5(2R,3S)—N-((3S)-1-(²H₃)Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide

In a 5 mL screw top vial was added Example 2 (50 mg, 0.092 mmol), cesiumcarbonate (60.1 mg, 0.184 mmol), and iodomethane-d₃ (6.88 μL, 0.111mmol) in DMF (2 mL) to give a suspension. The mixture was stirred atroom temperature under nitrogen overnight. LCMS showed the reaction wascomplete. The reaction mixture was dissolved in 2 ml of 1:1 DMF/AcOH andpurified by Preparative HPLC on Luna ODS 5 um 21.2×100 mm which waseluted with a 7 min gradient from 10% to 100% ACN/water 0.1% TFA to100%. The appropriate fractions was concentrated to afford Example 5 (35mg, 65%): HPLC: RT=3.04 min (CHROMOLITH® S5 ODS column 4 6×50 mm, 10-90%aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min,monitoring at 220 nm); MS (ES): m/z=599 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d₆) δ ppm 9.55 (1H, d, J=7.5 Hz), 7.71-7.80 (1H, m), 7.67 (2H, d,J=8.3 Hz), 7.51-7.60 (3H, m), 7.42-7.49 (2H, m), 7.30-7.39 (2H, m), 7.15(1H, s), 5.30 (1H, d, J=7.3 Hz), 2.75-2.87 (1H, m), 2.40-2.48 (1H, m),2.04-2.31 (4H, m), 1.46-1.76 (4H, m).

Examples 6 to 11

The compounds listed below were prepared according to the generalsynthetic procedure described in Example 1, using the appropriatebenzodiazepinone obtained by methods known to one skilled in the art,for example, Carter, M. C. et al., J. Med. Chem., 49:2311-2319 (2006).

TABLE 6

HPLC Ex. Ret Time No. Y Z Compound Name [M + H]⁺ (min)  6 H Cl(2R,3S)-N-((3S)-7-chloro-1-methyl-2-oxo- 591 17.21 ^(a)5-phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl) succinamide  7 OCH₃ H(2R,3S)-N-((3S)-8-methoxy-1-methyl-2- [M − H] = 585 15.86 ^(a)oxo-5-phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide  8 F H(2R,3S)-N-((3S)-8-fluoro-1-methyl-2-oxo-5- 575 10.317 ^(b)phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl) succinamide  9 H OCH₃(2R,3S)-N-((3S)-7-methoxy-1-methyl-2- 587 15.92 ^(a)oxo-5-phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide 10 H F(2R,3S)-N-((3S)-7-fluoro-1-methyl-2-oxo-5- 575 16.15 ^(a)phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl) succinamide 11 Cl H(2R,3S)-N-((3S)-8-chloro-1-methyl-2-oxo- 591 17.58 ^(a)5-phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl) succinamide ^(a) Xbridge Phenyl (4.6 × 150 mm), 3.5micron; 1 mL/min flow rate; gradient 10-100% B over 30 min (A: 0.05% TFAin water/CH₃CN (95:5), B: 0.05% TFA in water/CH₃CN (5:95) @ 220 and 250nm); 30 min run. ^(b) Xbridge Phenyl (4.6 × 150 mm), 3.5 micron; 1mL/min flow rate; gradient 10-100% B over 15 min (A: 0.05% TFA inwater/CH₃CN (95:5), B: 0.05% TFA in water/CH₃CN (5:95) @ 220 and 250nm); 15 min run.

Examples 12 to 18

The compounds listed below were prepared according to the generalsynthetic procedure described in Example 1, using the appropriatebenzodiazepinone obtained by methods known to one skilled in the art,for example, Carter, M. C. et al., J. Med. Chem., 49:2311-2319 (2006).

TABLE 7

HPLC Ex. Ret Time No. X Y Z Compound Name [M + H]⁺ (min) 12 OCH₃ H H(2R,3S)-N-((3S)-9-methoxy-2-oxo- 573 14.741 ^(a)5-phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide 13 H OCH₃ H (2R,3S)-N-((3S)-8-methoxy-2-oxo-[M − H] = 571  9.38 ^(b) 5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3- trifluoropropyl)succinamide 14 H HOCH₃ (2R,3S)-N-((3S)-7-methoxy-2-oxo- [M − H] = 571  9.14 ^(b)5-phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide 15 OCH₃ CN H (2R,3S)-N-((3S)-8-cyano-9- 598 0.95^(c) methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)- 2,3-bis(3,3,3-trifluoropropyl)succinamide 16 Cl Cl H (2R,3S)-N-((3S)-8,9-dichloro-2- 611  2.095^(d)oxo-5-phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide 17 F H H (2R,3S)-N-((3S)-9-fluoro-2-oxo-5-561  2.698^(e) phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3- trifluoropropyl)succinamide 18 Cl H H(2R,3S)-N-((3S)-9-chloro-2-oxo-5- 577  1.982^(d)phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide ^(a) Xbridge Phenyl (4.6 × 150 mm), 3.5micron; 1 mL/min flow rate; gradient 10-100% B over 12 min (A: 0.05% TFAin water/CH₃CN (95:5), B: 0.05% TFA in water/CH₃CN (5:95) @ 220 and 250nm); 25 min run. ^(b) Xbridge Phenyl (4.6 × 150 mm), 3.5 micron; 1mL/min flow rate; gradient 10-100% B over 12 min (A: 0.05% TFA inwater/CH₃CN (95:5), B: 0.05% TFA in water/CH₃CN (5:95) @ 220 and 250nm); 15 min run. ^(c)LCMS: MeOH/H₂O/0.1% TFA, BEH C18 2.1 × 50 mm 1.7 u,2 min gradient, wavelength = 254 nm. ^(d)MeOH/H₂O/0.1% TFA, WatersSunfire C₁₈ 2.1 × 30 mm, 2 min gradient, wavelength = 254 nm.^(e)CHROMOLITH ® ODS 4.6 × 50 mm (4 min grad) eluting with 10-90%aqueous MeOH over 4 minutes containing 0.% TFA, 4 mL/min, monitoring at220 nm.

Example 19(2R,3S)—N-((3S)-2-Oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-3-(4,4,4-trifluorobutyl)-2-(3,3,3-trifluoropropyl)succinamide

Preparation 19A:(2R,3R)-3-(tert-Butoxycarbonyl)-7,7,7-trifluoro-2-(3,3,3-trifluoropropyl)heptanoicacid

Preparation 19A was prepared from Preparation 9A (674 mg, 2.59 mmol) andPreparation 1P (500 mg, 1.850 mmol) according to the alternate procedureshown for Preparation 1E. Obtained Preparation 19A (521 mg, 28%): MS(ES): m/z=379 [M−H]⁻.

Preparation 19B:(2R,3S)-3-(tert-Butoxycarbonyl)-7,7,7-trifluoro-2-(3,3,3-trifluoropropyl)heptanoicacid

Preparation 19B was prepared from Preparation 19A (198 mg, 0.521 mmol)according to the general procedure shown to enrich the mixture ofPreparation 1E and Preparation 1F. Obtained Preparation 19B (192 mg,98%): MS (ES): m/z=379 [M−H]⁻; ¹H NMR (500 MHz, CDCl₃) δ ppm 2.65-2.72(1H, m), 2.60 (1H, ddd, J=10.33, 8.81, 3.61 Hz), 2.19-2.30 (2H, m),2.06-2.16 (3H, m), 1.85-1.96 (1H, m), 1.70-1.81 (2H, m), 1.51-1.67 (3H,m), 1.47 (7H, s).

Preparation 19C: tert-Butyl(2S,3R)-6,6,6-trifluoro-3-(((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(4,4,4-trifluorobutyl)hexanoate

Preparation 19C was prepared from Preparation 19B (45.4 mg, 0.119 mmol)and Preparation 2A (30 mg, 0.119 mmol) according to the generalprocedure shown for Preparation 1I. Obtained Preparation 19C (82 mg,100%): HPLC: RT=3.475 min (CHROMOLITH® 5 u C18 4.6×30 mm (4 min grad)eluting with 10-90% aqueous MeOH over 4 minutes containing 0.1% TFA,monitoring at 220 nm); MS (ES): m/z=614 [M+H]⁺.

Preparation 19D:(2S,3R)-6,6,6-Trifluoro-3-(((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(4,4,4-trifluorobutyl)hexanoicacid

Preparation 19D was prepared from Preparation 19C (73 mg, 0.119 mmol)according to the general procedure shown for Preparation 1K. ObtainedPreparation 19D (80 mg, 100%) as a TFA solvate: HPLC: RT=2.926 min(CHROMOLITH® 5 u C18 4.6×30 mm (4 min grad) eluting with 10-90% aqueousMeOH over 4 minutes containing 0.1% TFA, monitoring at 220 nm).

Example 19

Example 19 was prepared from Preparation 19D (80 mg, 0.119 mmol)according to the general procedure shown for Example 1. After separationof the diastereoisomers, Example 19 (35 mg, 49%) was obtained. HPLC:RT=2.731 min (CHROMOLITH® 5 u C18 4.6×30 mm (4 min grad) eluting with10-90% aqueous MeOH over 4 minutes containing 0.1% TFA, monitoring at220 nm); MS (ES): m/z=557 [M+H]⁺; ¹H NMR (500 MHz, DMSO-d₆) δ ppm 10.82(1H, s), 9.42 (1H, d, J=7.21 Hz), 7.65 (1H, ddd, J=8.32, 7.07, 1.53 Hz),7.60 (1H, d, J=2.22 Hz), 7.49-7.57 (3H, m), 7.42-7.49 (2H, m), 7.29-7.35(2H, m), 7.20-7.28 (1H, m), 7.03 (1H, s), 5.23 (1H, d, J=7.21 Hz),2.70-2.79 (1H, m), 2.57-2.69 (1H, m), 2.39-2.47 (1H, m), 2.05-2.32 (3H,m), 1.50-1.67 (3H, m), 1.40-1.49 (1H, m), 1.24-1.39 (2H, m).

Example 20(2R,3S)—N1-((3S)-8-Methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-3-(4,4,4-trifluorobutyl)-2-(3,3,3-trifluoropropyl)succinamide

Example 20 was prepared by using a sequence of reactions as outlined forExample 19 using Preparation 19B instead of Preparation 1E. The mixtureof diastereoisomers obtained was separated via chiral HPLC to provideExample 20. HPLC RT=0.89 min. (BEH C18 2.1×50 mm, 1.7 u, 0 to 100 B in 1min with 0.5 min hold time, Flow rate=1 ml/min, detection at 254 nm,Solvent A: 100% water/0.1% TFA; Solvent B: 100% ACN1/0.1% TFA). MS (ES):m/z=587.2 [M+H]⁺.

Example 21(2R,3S)—N-((3S)-9-((2-Methoxyethyl)amino)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide

Preparation 21A:(3-((4-Methoxybenzyl)(2-methoxyethyl)amino)-2-nitrophenyl)(phenyl)methanone

A mixture of (3-chloro-2-nitrophenyl)(phenyl)methanone (850 mg, 3.25mmol) and 2-methoxy-N-(4-methoxybenzyl)ethanamine (3171 mg, 16.24 mmol)was heated at 100° C. for 16 hours. The reaction mixture was partitionedbetween water (50 mL) and DCM (50 mL), extracted with DCM (3×50 mL),dried over Na₂SO₄, and purified using silica gel chromatography(stepwise gradient: 30 to 50% ethyl acetate/hexanes) to isolatePreparation 21A (780 mg, 57.1% yield) as a brown oil: LC/MS (PHENOMENEX®Luna 5 micron C18 4.6×30 mm, 0 to 100 B in 2 min with 1 min hold time,Flow rate=5 ml/min, detection at 254 nm, Solvent A: 10% methanol/90%water/0.1% TFA; Solvent B: 10% water/90% methanol/0.1% TFA) RT=2.32; MS(ES) m/z=443.10 [M+Na]⁺.

Preparation 21B:(2-Amino-3-((4-methoxybenzyl)(2-methoxyethyl)amino)phenyl)(phenyl)methanone

Preparation 21A (700 mg, 1.665 mmol), zinc (1089 mg, 16.65 mmol), andammonium chloride (891 mg, 16.65 mmol) in EtOH (40 mL) and water (20 mL)was heated to 90° C. for 5 minutes. The reaction mixture was filteredthrough CELITE®, partitioned between water/DCM, extracted 3×10 mL DCM,dried over Na₂SO₄, and concentrated to isolate Preparation 21B (580 mg,89% yield): LC/MS (PHENOMENEX® Luna 5 micron C18 4.6×30 mm, 30 to 100 Bin 4 min with 1 min hold time, Flow rate=5 ml/min, detection at 254 nm,Solvent A: 10% methanol/90% water/0.1% TFA; Solvent B: 10% water/90%methanol/0.1% TFA): RT=2.47 min; MS (ES): m/z=391.16 [M+H]⁺.

Preparation 21C: Benzyl9-((4-methoxybenzyl)(2-methoxyethyl)amino)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-ylcarbamate

Preparation 21C was prepared from Preparation 21B following the generalprocedure for Preparation 50D (38.6% yield): LC/MS (PHENOMENEX® Luna 5micron C18 4.6×30 mm, 30 to 100 B in 4 min with 1 min hold time, Flowrate=5 ml/min, detection at 254 nm, Solvent A: 10% methanol/90%water/0.1% TFA; Solvent B: 10% water/90% methanol/0.1% TFA) RT=2.42 min;MS (ES): m/z=579.22 [M+H]⁺.

Example 21

Example 21 was prepared from Preparation 21C by using the generalsequence of reactions as outlined for Example 1. The mixture ofdiastereoisomers obtained was separated via chiral HPLC to provideExample 21: LC/MS (PHENOMENEX® Luna 5 micron C18 4.6×30 mm, 30 to 100 Bin 4 min with 1 min hold time, Flow rate=5 ml/min, detection at 254 nm,Solvent A: 10% methanol/90% water/0.1% TFA; Solvent B: 10% water/90%methanol/0.1% TFA) RT=2.13 min; MS (ES): m/z=616.22 [M+H]⁺.

Comparative Compounds 22 to 25

Comparative Compounds 22 to 25 can be prepared according to theprocedures described in U.S. Pat. No. 7,053,084 for Examples 8, 12a, 38,and 45a, respectively.

Comparative Compound U.S. Pat. No. 7,053,084 Structure 22 Ex. 8

23 Ex. 12a

24 Ex. 38

25 Ex. 45a

Example 26 Pharmaceutical Formulation Comprising(2R,3S)—N-((3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide

An injection drug product was formulated comprising2R,3S)—N-((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide,Example 1, as a single-use, ready-to-use (RTU) sterile solution forintravenous (IV) administration using 50:50 (v/v) combinations ofpurified polyoxyethylated castor oil (a surfactant) and dehydratedalcohol (a solvent). The drug product was a clear to slightly hazy(opalescent), colorless to pale yellow sterile solution, stored in 5-mLType I flint glass vials, closed with 20-mm stoppers, and sealed with20-mm aluminum seals. The concentrated formulation can be diluted priorto administration with commonly used intravenous diluents, such asNormal Saline (NS) or 5% Dextrose, to provide a physiologicallyacceptable diluted product.

TABLE 11 Concentrated Pharmaceutical Composition Quantity Component(mg/mL) Example 1 active pharmaceutical ingredient 1.2 PurifiedPolyoxyethylated solubilizer 0.5 Castor Oil Dehydrated Alcohol solvent0.5 Purified Polyoxyethylated Castor Oil: CREMOPHOR (BASF Corp.)

The concentrated pharmaceutical formulation was found to be stable uponstorage at 25° C./60% relative humidity, 40° C./75% relative humidity,and 50° C. for a period of three months. Also, a photo-stability study(HIL/UVA) indicated that the product did not need to be protected fromlight. The concentrated formulation of Example 1 had long-term shelfstability including chemical and physical stability.

Prior to IV administration, the concentrated pharmaceutical formulationwas diluted with Normal Saline (NS) or 5% Dextrose in Water (D5W) toconcentrations between 0.01 mg/mL and 0.06 mg/mL. Theuse-time/compatibility results indicated that the product diluted in NSor D5W to concentrations in the range from 0.01 mg/mL to 0.06 mg/mL wascompatible with non-PVC, non-DEHP IV infusion bags. The results showedessentially no changes through 24 hours of storage at 2° C. to 8° C. orroom temperature/room light (25° C. and approximately 5000 lux).

Biological Assays

The pharmacological properties of the compounds of this invention may beconfirmed by a number of biological assays. The exemplified biologicalassays, which follow, have been carried out with compounds of theinvention.

Notch-CBF1 Transactivation Assay

The Notch-CBF1 (C-promoter binding factor I) cell based transactivationassay is based on the ability of the released Notch intracellular domainfragments (NICDs) to function as transcription factors in conjunctionwith CBF1 and other nuclear factors. Luciferase assays were used tomeasure the antagonism of Notch-CBF1 transcriptional activity. HeLacervical cancer cells are transiently co-transfected with pcDNA3.1/Hygroplasmids containing truncated Notch 1, Notch 2, Notch 3, or Notch 4receptors and a PGL3 luciferase reporter vector containing 4 copies ofCBF1 binding site. The cells were then tested for Notch-CBF1 activity inthe absence or presence of test compounds. HeLa cells, maintained inDMEM (high glucose with HEPES), 1× glutamine/penicillin/streptomycin and10% Fetal Bovine serum, were transiently transfected in a T175 Flask(4.5×10⁶ cells/flask) using the Monster Transfection Kit (Minis#MIR2906) according to manufacturers specifications. Table 12 denotesrespective DNA quantity for the transfections.

TABLE 12 DNA (μg) CBF1 (μg) Vector (μg) Total DNA (μg) human Notch 1 614.4 15.6 36.0 human Notch 2 2 14.4 19.6 36.0 human Notch 3 0.3 14.421.3 36.0 human Notch 4 4 14.4 17.6 36.0

Six hours post-transfection, cells were trypsinized and plated into a384-well black Poly-D-lysine coated tissue culture plate at a density of5×10³ cells/well in 95 μl, assay media (DMEM (high glucose with HEPES),1× glutamine/penicillin/streptomycin, 0.0125% BSA, 1× non-essentialamino acids). Assay media (5 mL) containing test compounds in finalconcentrations ranging from 5 μM to 8.4×10⁻⁵ μM (3 fold serialdilutions) were added to the cells and the cell plates were thenincubated for 18 hours at 37° C. and 5% CO₂. Control wells containedDMSO vehicle (total counts) or 0.5 μM of an in-house small moleculeinhibitor (background counts). Duplicates were used for each sample.Luciferase activity was measured after a 20-minute incubation with 50 μlSTEADY-GLO® luciferase reagents according to manufacturer'sspecifications (Promega, Cat. #: E2550) and analyzed by Envision platereader (PerkinElmer, Boston, Mass.).

The antagonist effect of compounds was expressed as 100×[1−(averagesample−average background)/(average total−average background)] wheresample is the luciferase activity in the presence of test compound,background is equal to the luciferase activity in the presence of thesmall molecule inhibitor control and the total is signal induced in DMSOwells. Data was plotted using a four parameter logistic fit equation andthe IC₅₀ value was defined as the concentration of compound thatinhibited 50% of the luciferase activity.

Table 13 below lists the Notch 1 and Notch 3 IC₅₀ values for Examples 1to 21 of this invention and Comparative Compounds 22 to 25 measured inthe Notch-CBF1 Transactivation Assay hereinabove. The results in Table13 were rounded to 2 digits. The compounds of the present invention, asexemplified by the Examples 1 to 21 showed Notch 1 values of 6.6 nM orless and Notch 3 IC₅₀ values of 13 nM or less.

TABLE 13 Notch 1 Notch 3 Example (IC₅₀, nM) (IC₅₀, nM)  1 1.6 3.4  2 1.73.3  3 3.1 4.7  4 1.5 2.5  5 1.2 5.9  6 6.5 10  7 1.5 2.8  8 4.9 8.1  94.4 8.2 10 2.9 4.6 11 1.3 2.0 12 2.5 4.2 13 2.1 3.8 14 5.2 13 15 12 1616 4.2 6.4 17 3.6 7.1 18 0.53 2.3 19 1.3 3.8 20 2.9 4.2 21 1.5 4.2Comparative Compound 22 64 48 Comparative Compound 23 42 75 ComparativeCompound 24 5.1 13 Comparative Compound 25 12 12

High Throughput (HT) Metabolic Stability Panel

Compounds administered parenterally enter the blood stream and undergoone or more passes through the liver. Compounds that are not readilymetabolized by the liver can be administered at therapeuticallyeffective plasma levels for therapeutically effective periods of time.

Orally administered compounds typically are absorbed through theintestinal walls into the blood stream and undergo a first pass throughthe liver. Compounds that are not readily metabolized in this first passthrough the liver can be distributed to other areas of the body intherapeutically effective amounts.

The metabolic stability assay evaluated CYP-mediated metabolic stabilityin vitro using human, rat, mouse, dog, and/or monkey microsomes after aten-minute incubation. Each compound was tested in duplicate.

The results of these assays were expressed as the fraction of parentcompound remaining in the reaction mixture after a ten-minute incubation(Percent Remaining). In general, these results were used to evaluateonly the extent of CYP-mediated, or NADPH-dependent, metabolism of thetest compound. When the compound was significantly metabolized (<40-50%remaining), this indicated high clearance of the compound in vivo due toCYP-mediated metabolism. However, if the compound demonstrated moderate(50-80%) or low (>85%) metabolism in these in vitro assays, highclearance was still possible in vivo via other metabolism andelimination pathways.

The percent remaining results of these assays was predictive of compoundclearance in vivo, assuming that CYP-mediated metabolism was apredominant elimination pathway. In different microsomal species, theranges of results were approximately as shown in Table 14.

TABLE 14 Metabolic Stability - Result Interpretation GuidelinesCYP-Mediated Percent Remaining after 10 minutes Clearance Human RatMouse Dog Monkey Low >90 >85 >85 >90 >85 Medium 60-90 40-85 50-85 55-9040-85 High <60 <40 <50 <55 <40

Table 15 below lists the CYP-mediated metabolic stability for Examples 1to 21 of this invention and Comparative Compounds 22 to 25 measured inthe human and mouse metabolic stability assays. The results in Table 15were rounded to 2 digits. In the liver microsome assays, a value of 0%remaining indicated complete CYP-mediated metabolism of a test compound,and a value of 100% indicated no detectable CYP-mediated metabolism of atest compound. The compounds of the present invention, as exemplified byExamples 1 to 21 had metabolic stability values of 80% or greaterremaining for human liver microsomes (HLM) and 72% or greater remainingfor mouse liver microsomes (MsLM). In contrast, Comparative Compounds 22to 25 had metabolic stability values of 39% or less remaining for humanliver microsomes and 15% or less remaining for mouse liver microsomes.

TABLE 15 0.5 μM HLM 0.5 μM MsLM Example (% Remaining) (% Remaining)  197 91  2 88 86  3 84 91  4 100 91  5 100 98  6 100 100  7 93 72  8 96100  9 100 87 10 96 98 11 97 98 12 100 100 13 100 97 14 100 95 15 100 9316 80 91 17 100 96 18 100 100 19 96 97 20 100 100 21 98 82 ComparativeCompound 22 39 15 Comparative Compound 23 19 9.0 Comparative Compound 2421 13 Comparative Compound 25 2.7 0.18

The compounds of the present invention (Examples 1 to 21) have beencompared to the Comparative Compounds 22 to 25 disclosed in U.S. Pat.No. 7,456,172, and have been found to be especially advantageous. Thecompounds of the present invention had the surprising advantage of thecombination of activity as inhibitors of Notch 1 and Notch 3 andsuperior metabolic stability to liver microsomes. As shown in Tables 13and 15, in the reported tests, Examples 1 to 21 of this invention hadNotch 1 IC₅₀ values of 6.6 nM or less and Notch 3 IC₅₀ values of 13 nMor less; and metabolic stability values of 80% or greater remaining forhuman liver microsomes (HLM) and 72% or greater remaining for mouseliver microsomes (MsLM). In contrast, in similar tests, the ComparativeCompounds 22 to 25 had Notch 1 IC₅₀ values of 5.1 nM or greater andNotch 3 IC₅₀ values of 13 nM or greater; and metabolic stability valuesof 39% or less remaining for human liver microsomes and 15% or lessremaining for mouse liver microsomes.

Methods and Materials

Incubation with Liver Microsomes

Test compound was received as a 3.5 mM stock solution in 100 percentDMSO. The test compound was diluted to create a 50 μM acetonitrile (ACN)solution containing 1.4% DMSO, which was then used as a 100× stock forincubation with microsomes. Each compound was tested in duplicateseparately in each of three species in the Metabolic Stability-Human,Rat, and Mouse assay suite or as individual species in the MetabolicStability-Dog or Metabolic Stability-Monkey suites. Compound, NADPH, andliver microsome solutions were combined for incubation in three steps:

1. 152 μl of liver microsome suspension, protein concentration of 1.1mg/ml in 100 mM NaP_(i), pH 7.4, 5 mM MgCl₂ buffer, was pre-warmed at37° C.

2. 1.7 μl of 50 μM compound (98.6% ACN, 1.4% DMSO) was added to the sametube and pre-incubated at 37° C. for 5 minutes.

3. The reaction was initiated by the addition of 17 μl of pre-warmed 10mM NADPH solution in 100 mM NaP_(i), pH 7.4.

The reaction components were mixed well, and 75 μl of the reactionmixture was immediately transferred into 150 μl quench/stop solution(zero-time point, T₀). Reactions were incubated at 37° C. for 10 minutesand then an additional 75 μl aliquot was transferred into 150 μl quenchsolution. Acetonitrile containing 100 μM DMN (a UV standard forinjection quality control), was used as the quench solution to terminatemetabolic reactions.

Quenched mixtures were centrifuged at 1500 rpm (˜500×g) in an ALLEGRA®X-12 centrifuge, SX4750 rotor (Beckman Coulter Inc., Fullerton, Calif.)for fifteen minutes to pellet denatured microsomes. A volume of 90 μl ofsupernatant extract, containing the mixture of parent compound and itsmetabolites, was then transferred to a separate 96-well plate forUV-LC/MS-MS analysis to determine the percent of parent compound thatremained in the mixture.

TABLE 16 Metabolic Stability Assay - Reaction Components FinalConcentration in the Metabolic Reaction Components Stability AssayCompound (Substrate) 0.5 μM NaPi Buffer, pH 7.4 100 mM DMSO 0.014%Acetonitrile 0.986% Microsomes (human, rat, mouse) (BD/Gentest) 1 mg/mlprotein NADPH 1.0 mM MgCl₂ 5.0 mM 37° C. Incubation time 0 minutes and10 minutes Quench/Stop Solution (ACN + 100 μM DMN) 150 μl Sample ofReaction 75 μl Sedimentation of Denatured Microsomes 15 minutes UV-LC/MSanalysis of supernatant 0.17 μM

Sample Analysis—Instrumentation

HPLC: Pump—Thermo Surveyor; Autosampler—CTC/LEAP HTS; UV detector—ThermoSurveyor PDA plus; Column—Varian C18, 3 μm, 2×20 mm with a 0.5 μmin-line filter; Mobile Phase for structural integrity pre-analysis: (A)98% water, 2% acetonitrile with 10 mM ammonium acetate; (B) 10% water,90% acetonitrile with 10 mM ammonium acetate; Mobile Phase for reactionsample analysis: (A) 98% water, 2% acetonitrile with 0.1% formic acid;(B) 2% water, 98% acetonitrile with 0.1% formic acid; (C) 0.1% ammoniumhydroxide in water; (D) 0.1% ammonium hydroxide in acetonitrile.

Mass Spectrometer Thermo TSQ Quantum Ultra triple-quadrapole massspectrometer;

Sample Analysis Structural Integrity Pre-Analysis.

The Metabolic Stability structural integrity pre-analysis was used toassess the purity of compounds being assayed. Compounds were received in96-well plates as 57 μl of a 3.5 mM DMSO solution. The 3.5 mM compoundDMSO stock solutions were diluted 18-fold with a solution containingequal volumes of acetonitrile, isopropanol, and MilliQ-H₂O. Theresulting solutions (200 μM) were analyzed for structural integrity byLC-UV/MS on a Thermo LCQ Deca XP Plus ion trap mass spectrometer, usinga Waters XBridge C18, 5 μm, 2×50 mm column with a Waters Sentry 2.1 mmguard column, and the LC conditions described in the table below, with a5 μl injection and a flow rate of 1 ml/min. The acquired data reflectedpurity by UV absorbance at 220 nm. Only results for those compounds withpurity greater than 50% were reported.

TABLE 17 Metabolic Stability - Structural Integrity Gradient GradientTime (min) A % B % 0.00 100 0 4.00 0 100 5.00 0 100 5.10 100 0 6.00 1000

Sample Analysis—Incubated Samples

MS/MS condition optimization was conducted on a Thermo TSQ Quantumtriple-quadrapole mass spectrometer equipped with a heated-electrospray(H-ESI) source by automated infusion to obtain the SRM transitions andtheir corresponding collision energy values. Compound solutions at aconcentration of 20 μM in 1:1 methanol:water were infused at a flow rateof 90 μL/min, then combined with the mobile phase at a flow rate of 50μL/min before being introduced into the source. All compounds wereoptimized first using mobile phase A and B (50% A and 50% B), and ifnecessary, using mobile phase C and D (also with a 50:50 composition).The optimized parameters, including polarity, SRM transition andcollision energy, were stored in a Microsoft Access database.

The mass spectrometric conditions obtained from automated infusion wereused to analyze incubation samples from the Metabolic Stability assay.The injection volume was 5 μl and the flow rate was 0.8 ml/min. Thegradient used was shown in the table below. All samples were injectedwith the gradient using mobile phase A and B first. If necessary (forinstance, for chromatographic reasons), samples were re-injected withthe same gradient, but using mobile phase C and D. All LC-MS/MS analysisparameters were captured electronically in the raw data files.

TABLE 18 Metabolic Stability - Sample Analysis Gradient Gradient Time(min) A % (or C %) B % (or D %) 0.00 95 5 0.20 95 5 0.30 0 100 1.05 0100 1.10 95 5 1.50 95 5

Data Analysis

Peak integration was performed with the XCALIBUR® software. The percentremaining calculation was performed by comparing the LC-MS/MS peak areasfrom the T_(10minute) samples to those from the T_(10minute) samples foreach compound.

Quality Control

A set of three compounds was tested along with the test compound in eachassay plate. Data was accepted and uploaded only if the results forthese control compounds fall into the expected ranges shown below.

TABLE 19 Metabolic Stability Assay - Control Compound Values byMicrosome Species Average Percent Remaining ± SD Compound Human RatMouse Dog Monkey Nefazodone  0.4 ± 0.4 0.7 ± 0.6 0.4 ± 0.3 0.4 ± 0.4 0.6± 0.5 Verapamil 13.3 ± 3.5 4.4 ± 2.1 13.0 ± 4.2  5.6 ± 1.8 0.5 ± 0.5Carbamezepine 96 ± 6 84 ± 9  90 ± 10 81 ± 7  89 ± 13 SD = StandardDeviation

Metabolic Stability Half-Life Panel

The rate of metabolism and half-life determined in vitro in human oranimal liver microsomes was used to determine intrinsic clearance(CL_(int)) and hepatic clearance (CLh,b) of a compound. These parameterswere useful for predicting in vivo human clearance, which defines thelevel of drug exposure in vivo (Obach et al., J. Pharmacol. Exp. Ther.,283:46-58 (1997); Obach, Drug Metab. Dispos., 27:1350-1359 (1999)).

The metabolic stability half-life assay panel evaluates the time-courseand the rate of CYP-mediated (NADPH-dependent) metabolism in vitro inhuman, rat, mouse, dog and monkey microsomes. The time course spans a 45minute incubation, and includes 0, 5, 10, 15, 30, and 45 minutetime-points, at each of which the amount of test compound remaining inthe mixture was measured.

Result Interpretation Guideline

The results of these assays were expressed as a half-life (T_(1/2),min), and the fraction of parent compound remaining in the reactionmixture at each time-point (Percent Remaining) was also reported. Ingeneral, these results should be used to evaluate only the extent ofCYP-mediated, or NADPH-dependent, metabolism of the test compound. Whenthe compound was significantly metabolized (T_(1/2)<8-14 min), thisindicated high clearance in vivo due to CYP-mediated metabolism.However, if the compound demonstrated moderate (50-80%) or low (>85%)metabolism in these in vitro assays, high clearance was still possiblein vivo via other metabolism and elimination pathways.

The results of these assays was predictive of compound clearance invivo, assuming that CYP-mediated metabolism was a predominantelimination pathway. In different microsomal species, the ranges ofresults were approximately as shown in the following table:

TABLE 20 Metabolic Stability Half-Life - Result InterpretationGuidelines CYP-Mediated T_(1/2), minutes Clearance Human Rat Mouse DogMonkey Low >70 >40 >50 >65 >40 Medium 14-70 8-40 10-50 12-65 8-40 High<14  <8 <10 <12  <8

Methods and Materials

Liver microsomes were purchased from BD-Biosciences (Woburn, Mass.) andNADPH from AppliChem Inc; all other reagents were obtained from Sigma.

Incubation with Liver Microsomes

Test compound was received as a 3.5 mM stock solution in 100 percentDMSO. The test compound was diluted to create a 50 μM acetonitrile (ACN)solution containing 1.4% DMSO, which was then used as a 100-fold stockfor incubation with microsomes. Each compound was tested in human, rat,mouse, dog and monkey liver microsomes. Compound, NADPH and livermicrosome solutions were combined for incubation in three steps:

1. 450 μl of liver microsome suspension, protein concentration of 1.1mg/ml in 100 mM NaP_(i), pH 7.4, 5 mM MgCl₂ buffer, was pre-warmed at37° C.

2. 5 μl of 50 μM compound (98.6% ACN, 1.4% DMSO) was added to the sametube and pre-incubated at 37° C. for 5 minutes.

3. The reaction was initiated by the addition of 50 μl of pre-warmed 10mM NADPH solution in 100 mM NaP_(i), pH 7.4.

Reaction components were mixed well, and 65 μl were immediatelytransferred into 130 μl quench/stop solution (zero-time point, T₀).Reactions were incubated at 37° C. for 5, 10, 15, 30 and 45 minutes andat each time-point a 65 μl aliquot was transferred into 130 μl of quenchsolution. Acetonitrile containing Internal Standard (100 ng/ml), wasused as the quench solution to terminate metabolic reactions.

Quenched mixtures were centrifuged at 1500 rpm (˜500×g) in an ALLEGRA®X-12 centrifuge, SX4750 rotor (Beckman Coulter Inc., Fullerton, Calif.)for fifteen minutes to pellet denatured microsomes. A volume of 90 μl ofsupernatant extract, containing the mixture of parent compound and itsmetabolites, was then transferred to a separate 96-well plate forLC/MS-MS analysis to determine the percent of parent compound that wasremaining in the mixture.

TABLE 21 Metabolic Stability Half-Life Assays - Reaction ComponentsFinal Concentration in the Reaction Components Metabolic Stability AssayCompound (Substrate) 0.5 μM NaPi Buffer, pH 7.4 100 mM DMSO 0.014%Acetonitrile 0.986% Microsomes (human, rat, 1 mg/ml protein mouse)(BD/Gentest) NADPH 1.0 mM MgCl₂ 5.0 mM 37° C. Incubation time 0, 5, 10,15, 30, and 45 minutes Quench/Stop Solution 130 μl (ACN + 100 μM DMN)Sample of Reaction 65 μl Sedimentation of Denatured 15 minutesMicrosomes

Sample Analysis—Instrumentation

HPLC: Pump—Shimadzu LC-20 AD series binary pumps; Autosampler—CTC/LEAPHTS.

The exemplified compounds of the invention showed the surprisingadvantage of low clearance due to CYP-mediated metabolism in both thehuman (HLM) and mouse (MsLM) metabolic stability assays. The compoundsof the present invention, as exemplified by Examples 1-2, 5-7, 9-14, 16,18, 21-23, and 26, had percent remaining values in the range of 60% to100% for the human liver microsome assay, and 25% to 100% for the mouseliver microsome assay. In contrast, Comparative Compounds 60-61 hadpercent remaining values of 7.0% or less in both the human and mouseliver microsome assays. Comparative Compounds 61-62 showed highclearance in both the human and mouse metabolic stability assays,indicating that the compounds were removed by CYP-mediated metabolism inthe liver.

Human Tumor Xenograft Models in Mice

All rodents were obtained from Harlan Sprague Dawley Co. (Indianapolis,Ind.), and maintained in an ammonia-free environment in a defined andpathogen-free colony. All mice were quarantined approximately 1 weekprior to their use for tumor propagation and drug efficacy testing. Micewere fed food and water ad libitum. The animal care program ofBristol-Myers Squibb Pharmaceutical Research Institute is fullyaccredited by the American Association for Accreditation of LaboratoryAnimal Care (AAALAC). All experiments were performed in accordance withBristol-Myers Squibb (BMS) animal test methods and guidelines.

Tumor xenografts were grown and maintained subcutaneously (SC) inimmunocompromized balb/c nu/nu nude or NOD-SCID mice (Harlan SpragueDawley). Tumors were propagated as subcutaneous transplants in theappropriate mouse strain (Table 22) using tumor fragments obtained fromdonor mice.

TABLE 22 Histological Types and Host Mouse Strain/Gender Requirement forthe Propagation of Various Human Tumor Xenografts in Mice Tumor TypeHistology Mouse Strain Sex TALL-1 ALL NOD-SCID female HPB-ALL ALLNOD-SCID female ALL-SIL ALL NOD-SCID female MDA-MB-157 breast NOD-SCIDfemale MDA-MB-468 breast NOD-SCID female PAT-34 ovarian nude femalePAT-50 ovarian nude female PAT-26 pancreas nude female PAT-27 pancreasnude female

Preclinical Chemotherapy Trials

The required numbers of animals needed to detect a meaningful responsewere pooled at the start of the experiment and each was given asubcutaneous implant of a tumor fragment (˜20 mg) with a 13-gaugetrocar. Tumors were allowed to grow to the pre-determined size window(tumors outside the range were excluded) and animals were evenlydistributed to various treatment and control groups. There weretypically 8 mice per treatment and control groups, with the exception ofexperiments conducted in the SAL-IGF (this is not included in Table 22)tumor model, in which there were typically 5 mice per treatment andcontrol group. Treatment of each animal was based on individual bodyweight. Treated animals were checked daily for treatment relatedtoxicity/mortality. Each group of animals was weighed before theinitiation of treatment (Wt₁) and then again following the lasttreatment dose (Wt₂). The difference in body weight (Wt₂−Wt₁) provides ameasure of treatment-related toxicity.

Tumor response was determined by measurement of tumors with a calipertwice a week, until the tumors reached a predetermined “target” size of0.5 gm or 1 gm depending on the tumor type. Tumor weights (mg) wereestimated from the formula:

Tumor weight=(length×width²)÷2

Tumor response criteria are expressed in terms of tumor growthinhibition (% TGI). Tumor growth delay is defined as the difference intime (days) required for the treated tumors (T) to reach a predeterminedtarget size compared to those of the control group (C). For thispurpose, the tumor weight of a group is expressed as medium tumor weight(MTW).

Tumor growth inhibition is calculated as follows:

${\% \mspace{14mu} {Tumor}\mspace{14mu} {Growth}\mspace{14mu} {Inhibition}} = \frac{\left( {1 - {\frac{T_{t}}{T_{0}}*\frac{C_{0}}{C_{t}}}} \right)}{\left( {1 - \frac{C_{0}}{C_{t}}} \right)}$

where,

C_(t)=Median control tumor size at end of treatment

C₀=Median control tumor size at treatment initiation

T_(t)=Median tumor size of treated group at end of treatment

T₀=Median tumor size of treated group at treatment initiation

Activity is defined as the achievement of durable tumor growthinhibition of 50% or greater (i.e., TGI≧50%) for a period equivalent toat least 1 tumor volume doubling time and drug treatment must be for aperiod equivalent to at least 2 tumor volume doubling time.

Tumor response was also expressed in terms of tumor growth delay (TGDvalue), defined as the difference in time (days) required for thetreated tumors (T) to reach a predetermined target size compared tothose of the control group (C).

Whenever possible, antitumor activity was determined at a range of doselevels up to the maximum tolerated dose (MTD) which is defined as thedose level immediately below which excessive toxicity (i.e., more thanone death) occurred. When death occurred, the day of death was recorded.Treated mice dying prior to having their tumors reach target size wereconsidered to have died from drug toxicity. No control mice died bearingtumors less than target size. Treatment groups with more than one deathcaused by drug toxicity were considered to have had excessively toxictreatments and their data were not included in the evaluation of acompound's antitumor efficacy.

Potential drug toxicity interaction affecting treatment tolerability isan important consideration in combination chemotherapy trials.Interpretation of combination therapeutic results must be based oncomparison of antitumor activity of the best possible response for thesingle agents versus the combination at comparably tolerated doses.Therefore, therapeutic synergism was defined as a therapeutic effectachieved with a tolerated regimen of the combined agents that exceededthe optimal effect achieved at any tolerated dose of monotherapy.Statistical evaluations of data were performed using Gehan's generalizedWilcoxon test. Statistical significance was declared at P<0.05.

Drug Administration

In in vitro studies, all agents were dissolved in 100% DMSO and seriallydiluted in media/10% fetal bovine serum. For administration of Notchinhibitors to rodents, two different excipients were used: [1] 94%Labrafil/5% ETOH/1% TW80 or [2] ETOH/TPGS/PEG300 (10:10:80). Notchinhibitors were typically administered orally on a schedule of QD×15, 10day-on-2 day-off, although other schedules had also been evaluated andshown to be efficacious. For example, dosing regimen consisting ofQD×12, 4 day-on-3 day-off was shown to be equally efficacious as QD×15,10 day-on-2 day-off.

In Vivo Antitumor Activity

The antitumor activity of Example 1 administered via the intravenousroute (IV) was evaluated in human tumor xenografts implanted in mice. Asshown in FIG. 6, Example 1 exhibited antitumor activity.

Table 23 below lists the antitumor activity of examples of thisinvention measured in the Human Tumor Xenograft Models in mice. Thecompounds of the present invention, as exemplified by Examples 1 and 2,showed antitumor activity with oral administration (PO).

TABLE 23 Schedule: QDx15, 10 Day-on-2 Day-off; Oral AdministrationAntitumor Activity Dose TALL1 MDA-MB-157 MDA-MB-468 Example (mg/kg)(LCK) (% TGI) (% TGI) 1 7.5-10 >4.7 89 78 2 24 2.4 85 87 QD - once dailyLCK—Log Cell Kill

Example 1 demonstrates broad-spectrum antineoplastic activity against awide array of human cancer xenografts grown in mice. Significantantitumor activity was demonstrated in 16 human cancer xenografts,including human T-cell acute lymphoblastic leukemia, breast carcinoma,pancreatic carcinoma, ovarian carcinoma, glioblastoma, non-small celllung carcinoma, colon carcinoma, osteogenic sarcoma, and neuroblastoma(Table 24).

TABLE 24 Antitumor Activity Tumor Histology (% TGI)^(a) TALL1 T-Cellacute lymphoblastic 112 leukemia Pat-24 pancreatic cancer 111 BT-474HER2+ breast cancer 96 Pat-26 pancreatic cancer 93 MDA-MB468 TN breastcancer 91 Pat-50 ovarian cancer 91 Pat-34 ovarian cancer 89 U-87glioblastoma multiforme (GBM) 82 MDA-MB157 TN breast cancer 81 Calu-6Non small cell lung cancer 81 HCT116 colon cancer 75 G292 osteogenicsarcoma 75 Pat-21/Abx R TN breast cancer (abx R) 73 MCF7estrogen-dependent breast cancer 73 SK-N-AS neuroblastoma 67 MCF7iestrogen-independent breast cancer 63 ^(a)All treatments were PO, QDx15,10 day-on-2 day-off, at dosages ranging from 5-10 mg/kg/adm.

Combination Chemotherapy

A series of studies were conducted to evaluate the combinability ofExample 1 with a number of anti-cancer agents including dasatinib,paclitaxel, Tamoxifen, dexamethasone, and carboplatin

I. Example 1 and Dasatinib

The human T-cell lymphoblastic leukemia was used to evaluate thecombined efficacy Example 1 and dasatinib. Dasatinib treatment aloneproduced an antitumor effect of 1.7 LCK (10 mg/kg/adm, QD×49, PO).Example 1 compound produced only modest activity of 0.1-0.5 LCK at doserange of 3.75-7.5 mg/kg. However, combination of the two agents producedsynergistic antitumor activity, yielding antitumor efficacy of >>2.6 LCKthat was significantly superior to dasatinib single agent alone(P<0.05). In addition, the combination regimen yielded complete response(CR) in 100% of mice, whereas none of the single agent produced CR(FIGS. 7-8 and Table 25).

TABLE 25 Antitumor Efficacy by Combined Chemotherapy with Example 1 andDasatinib in ALL-SIL T-cell Lymphoblastic Leukemia Treatment EfficacyExample 1 Dasatinib Tumor Dose^(a) Dose^(b) Growth Delay^(c) (mg/kg)(mg/kg) (LCK) (days) PR (%) CR (%) P 7.5  — 0.1 1.9 0 0 — 3.75 — 0.5 8.70 0 — — 10 1.7 30.2 0 0 1 7.5  10 >>2.6 44 0 100 <0.05 3.75 10 >>2.6 440 100 <0.05 ^(a)Regimen = PO, QD x3, weekly x7 ^(b)Regimen = PO, QD x49^(c)Target tumor size = 1000 mg

II. Example 1 and Paclitaxel

The antitumor efficacy of Example 1 in combination with paclitaxel wasevaluated in the MDA-MB-468 breast carcinoma. Example 1 as a singleagent produced 0.5-1.4 LCK at the dose range of 3.75-7.5 mg/kg/adm.Paclitaxel administered weekly at a dose of 12 mg/kg/adm yielded 0.5 LCK(FIGS. 9-10 and Table 26). The combination of Example 1 at thedose-range of 3.75-7.5 mg/kg/adm and paclitaxel produced 3.4-4.1 LCK ofantitumor effects which was significantly superior to single agentExample 1 compound alone (P=0.0006 and 0.0002, respectively).

TABLE 26 Antitumor Efficacy by Combined Chemotherapy with Example 1 andPaclitaxel in MDA-MB-468 Human Breast Carcinoma Treatment EfficacyExample 1 Paclitaxel Tumor Dose^(a) Dose^(b) Growth Delay^(c) (mg/kg)(mg/kg) (LCK) (days) PR (%) CR (%) P 7.5  — 1.4 21.2 0 0 1 3.75 — 0.57.8 0 0 — — 12 0.5 7.8 0 0 — 7.5  12 4.1 61.8 50 0 0.0002 3.75 12 3.451.2 0 0 0.0006 ^(a)Regimen = PO, QD x3, weekly x7 ^(b)Regimen = IV, Q7Dx6 ^(c)Target tumor size = 500 mg

III. Example 1 and Tamoxifen

The antitumor efficacy of Example 1 in combination with Tamoxifen wasevaluated in the ER receptor positive human breast carcinoma xenograftMCF7 grown in female nu/nu mice. Example 1 as a single agent producedtumor growth inhibition (TGI) of 43-58% at the dose range of 3.75-7.5mg/kg/adm with no CR or PR. Tamoxifen, administered at the MTD dose of20 mg/kg/adm, IP, Q2 DX12, produced % TGI of 78%, with no CR or PR. Thecombinations of Example 1 compound and Tamoxifen were clearlysynergistic producing % TGI of 101 and 99, respectively, at Example 1doses of 7.5 and 3.75 mg/kg/adm. Moreover, approximately 50% of micereceiving the combinations experienced tumor shrinkage, either as PR orCR (FIGS. 11-12 and Table 27).

TABLE 27 Antitumor Efficacy by Combined Chemotherapy with Example 1 andTamoxifen in the MCR7 Human Breast Carcinoma Treatment Efficacy Example1 Tamoxifen Tumor Growth Dose^(a) Dose^(b) Inhibition (mg/kg) (mg/kg)TGI^(c) PR (%) CR (%) P 7.5  — 58 0 0 0 3.75 — 43 0 0 — — 20 78 0 0 17.5  20 101 43 0 0.0012 3.75 20 99 43 14 0.0087 ^(a)Regimen = PO, QD x3,weekly x3 ^(b)Regimen = IP, Q2D x10 ^(c)Target tumor size = 500 mg

IV. Example 1 and Dexamethasone

The antitumor efficacy of Example 1 in combination with theglucocorticoid, dexamethasone, was evaluated in the human T-ALL leukemiaxenografts HPB-ALL grown in NOD-SCID mice. Example 1 as a single agentwas active in this model yielding 1.1 LCK at 7.5 mpk. Dexamethasone wasmodestly active as a single agent producing 0.7 LCK at its MTD of 7.5mpk. The combination of Example 1 and dexamethasone produced 1.9 LCK,significantly superior to either individual single agents alone (FIG. 13and Table 28).

TABLE 28 Antitumor Efficacy by Combined Chemotherapy with Example 1 andDexamethasone in HPB-ALL Human Acute Lymphoblastic Leukemia TreatmentEfficacy Example 1 Dexamethasone Tumor Dose^(a) Dose^(b) GrowthDelay^(c) PR (mg/kg) (mg/kg) (LCK) (days) (%) CR (%) P 7.5  — 1.1 9.5 00 0.0007 3.75 — 0.9 7.8 0 0 0.0007 — 7.5 0.7 5.8 0 0 0.0007 — 3.75 0.65.6 0 0 0.0007 3.75 7.5 1.9 16.5 0 0 1 ^(a)Regimen = PO, QD x3, weeklyx3 ^(b)Regimen = IP, QD x14 ^(c)Target tumor size = 3000 mg

V. Example 1 and Carboplatin

The antitumor efficacy of Example 1 in combination with carboplatin wasevaluated in the human ovarian teratocarcinoma xenograft PA-1 grown infemale nu/nu mice. Example 1 as a single agent produced 0.2 LCK at thedose of 1 mg/kg/adm. Carboplatin administered weekly at a dose of 90mg/kg/adm yielded 2.1 LCK (FIG. 14 and Table 29). The combination ofExample 1 at the dose of 1 mg/kg/adm and carboplatin produced >3.1 LCKof antitumor effects which was significantly superior to single agentExample 1 compound alone (P=0.004).

TABLE 29 Antitumor Efficacy by Combined Chemotherapy with Example 1 andCarboplatin in PA-1 Human Ovarian Teratocarcinoma Treatment Exam-Efficacy ple 1 Dexamethasone Tumor Dose^(a) Dose^(b) Growth Delay^(c) CRPR Cure (mg/kg) (mg/kg) (LCK) (days) (%) (%) (%) P 1 — 0.2 4 0 0 0 — —90 2.1 34.7 13 71 13 1 1 90 >3.1 >51.4 67 33 67 0.004 ^(a)Regimen = PO,QD x21 (1 mg/kg) ^(b)Regimen = IV, Q7D x3 ^(c)Target tumor size = 500 mg

Single Crystal X-Ray Diffractometry

The single crystal data were collected on a Bruker-AXS APEX2 CCD systemusing Cu Kα radiation (λ=1.5418 Å). Indexing and processing of themeasured intensity data were carried out with the APEX2 software programsuite. When indicated, crystals were cooled in the cold stream of anOxford cryo system during data collection. The structures were solved bythe direct methods and refined on the basis of observed reflectionsusing the SHELXTL. The derived atomic parameters (coordinates andtemperature factors) were refined through full matrix least-squares. Thefunction minimized in the refinements was Σ_(w)(|F_(o)|−|F_(c)|)². R isdefined as Σ∥F_(o)|−|F_(c)∥/Σ|F_(o)| whileR_(w)=[Σ_(w)(|F_(o)|−|F_(c)|)²/Σ_(w)|F_(o)|²]^(1/2) where w is anappropriate weighting function based on errors in the observedintensities. Typically, all the non-H atoms were refined anisotropicallyand all H-atoms other than those attached to N and O atoms werecalculated by geometrical methods and refined using a riding model.

X-Ray Powder Diffractometry

X-ray powder diffraction (PXRD) data were obtained using a Bruker GADDS(General Area Detector Diffraction System) manual chi platformgoniometer. Powder samples were placed in thin walled glass capillariesof 0.7 mm in diameter; the capillaries were rotated during datacollection. The sample-to-detector distance was kept at 17 cm. Data werecollected with Cu Kα radiation (λ=1.5418 Å) in the range 2.5<2θ<35° witha sample exposure time of 600 seconds.

1-20. (canceled)
 21. A compound of Formula (I):

wherein: R₁ is —CH₂CF₃ or —CH₂CH₂CF₃; R₂ is —CH₂CF₃, —CH₂CH₂CF₃, or—CH₂CH₂CH₂CF₃; R₃ is H or —CH₃; each R_(a) is independently F, Cl, —CN,—OCH₃, and/or —NHCH₂CH₂OCH₃; and z is 1 or
 2. 22. The compound accordingto claim 21 wherein: R₂ is —CH₂CF₃ or —CH₂CH₂CF₃.
 23. The compoundaccording to claim 21 wherein: z is
 1. 24. The compound according toclaim 23 selected from:(2R,3S)—N-((3S)-7-chloro-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(6);(2R,3S)—N-((3S)-8-methoxy-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(7);(2R,3S)—N-((3S)-8-fluoro-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(8);(2R,3S)—N-((3S)-7-methoxy-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(9);(2R,3S)—N-((3S)-7-fluoro-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(10);(2R,3S)—N-((3S)-8-chloro-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(11);(2R,3S)—N-((3S)-9-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(12);(2R,3S)—N-((3S)-8-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(13);(2R,3S)—N-((3S)-7-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(14);(2R,3S)—N-((3S)-9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(17); and(2R,3S)—N-((3S)-9-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide (18).
 25. The compound according to claim 21 wherein: z is2.
 26. The compound according to claim 25 selected from:(2R,3S)—N-((3S)-8-cyano-9-methoxy-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(15); and(2R,3S)—N-((3S)-8,9-dichloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide(16).